Warfarin inhibits the vitamin K dependent pathway that is necessary to synthesize functional coagulation factors II, VII, IX and X and the antithrombotic Proteins C and S. Vitamin K is the only specific antidote to warfarin. Even though IV administration of vitamin K begins to noticeably correct the INR within two hours after administration, plasma is often ordered for emergency reversal of warfarin induced anticoagulation. As documented in the May 2012 issue of the Clinical Laboratory Letter, the higher the INR the greater the correction per unit of plasma. Plasma transfusion is largely ineffective in correcting an INR of 2.2 or below.

In April 2013, FDA approved the use of Kcentra for the urgent reversal of acquired coagulation deficiency due to warfarin treatment among adult patients with acute major bleeding. In December, FDA extended its approval to include reversal of warfarin therapy for patients who require urgent, invasive medical procedures.

Kcentra is the brand name for a four factor Prothrombin Complex Concentrate which contains vitamin K dependent coagulation factors II, VII, IX and X and the antithrombotic Proteins C and S. Kcentra is indicated for emergent reversal of acquired coagulation factor deficiency induced by warfarin in adult patients with acute major bleeding or who need urgent surgery or another invasive procedure. Repeat dosing with Kcentra is not supported by clinical data and is not recommended.

Kcentra reduces INR and increases vitamin K dependent coagulation factor levels more rapidly than plasma and with much smaller volume. For example, a typical dose of 2000 units of Kcentra is reconstituted to a volume of 80 mL and is given IV push. Four bags of plasma is approximately 1000 mL and must be administered much more slowly to prevent circulatory overload. Additional time is required to thaw frozen plasma and determine a patient’s blood type. KCentra is supplied as a lyophilized powder that is reconstituted with 20 to 40 mL of sterile water at the bedside. The actual potency of Factors II, VII, IX and X, Proteins C and S in each vial is printed on each carton, but for convenience it is supplied as 500 and 1000 unit vials based on Factor IX concentration. Saint Luke’s Health Systems’ order sets for intracerebral hemorrhagic stroke and subarachnoid hemorrhagic stroke include a simplified dosing regimen based on body weight in kilograms and INR level. Generally, doses range between 2000 units for a person with an INR less than 4.0 and body weight <80 kg to 3500 units for a person with an INR of 4.0 or greater and body weight of 80 kg or higher. These doses correspond to 20-40 units of Factor IX per kg of body weight. Forty two patients who were anticoagulated with warfarin and developed intracerebral hemorrhage, subarachnoid hemorrhage or subdural hemorrhage have been treated with Kcentra since November 2013. Pretreatment INR ranged from 1.8 to 19.0. In each case Kcentra corrected the INR to 1.3 or less almost immediately upon infusion.

Vitamin K should be administered concurrently to patients receiving Kcentra to maintain vitamin K-dependent clotting factor levels because coagulation Factor VII has a short in vivo half life of 7 hours and Factor IX has a large volume of distribution.

Kcentra is contraindicated in patients with:

• Known anaphylactic or severe systemic reactions to any components in Kcentra including heparin, coagulation factors and human albumin
• Disseminated intravascular coagulation
• Heparin-induced thrombocytopenia

The most common adverse reactions observed in subjects receiving Kcentra are headache, nausea/vomiting, arthralgia, and hypotension. Arterial and venous thromboembolic complications have been reported in patients receiving Kcentra. Its use may not be suitable in patients with a history of thromboembolic events in the prior 3 months.

Kcentra is much more expensive than plasma. The medical record must clearly document the medical need to urgently correct warfarin induced anticoagulation to increase the likelihood of reimbursement.

Cytomegalovirus (CMV) is a DNA virus of the herpesvirus family. Transfusion acquired CMV is of little concern in immunocompetent individuals, but can be a serious problem in immunocompromised patients. In the latter group of patients, CMV transmission can result in pneumonitis, hepatitis, gastroenteritis, chorioretinitis, or disseminated disease. CMV negative blood components are indicated for fetal and intrauterine transfusions, low birth weight premature infants born to CMV seronegative mothers and CMV negative recipients of organ, peripheral blood stem cell or bone marrow transplants from CMV negative donors.

 Between 50 and 80% of the US population has been infected with CMV. Traditionally, blood banks test donors for antibodies to CMV to try to prevent viral transmission to immunocompromised patients. Labelling a unit as seronegative indicates that the unit does not contain detectable antibodies against CMV, but does not mean the unit cannot transmit CMV. A donor with a recent infection can harbor virus in their plasma or white blood cells even though they test negative for antibodies. The window period for CMV infection is estimated to be 6 to 8 weeks. As with all laboratory tests, there is the possibility of a false-negative antibody test in an infected donor.

In established infections, CMV resides in a small minority of monocytes. This means that risk of CMV transmission can be greatly diminished by leukocyte reduction of donor units, which removes at least 99.9% of leukocytes. Studies have estimated that fewer than 25 potentially infected monocytes remain following leukocyte reduction by either filtration or apheresis. During the viremic phase of an acute infection, some virus may circulate in the plasma and not be removed by leukocyte reduction. Neither antibody testing or leukocyte reduction will prevent transmission of CMV in this scenario.

In 1995, the Bowden study compared CMV transmission rates in bone marrow transplant recipients receiving either CMV seronegative or filtered blood products (Blood, Vol 86, No 9, 1995: pp 3598-3603). This study did not detect any significant difference in CMV transmission between either group.

Since 1997, AABB has considered leukocyte reduced blood to be a CMV safe product that is equivalent to CMV seronegative units for preventing CMV transmission. The Circular of Information for the Use of Human Blood and Blood Components (prepared jointly by AABB, American Red Cross, America’s Blood Centers and Armed Services Blood Program and recognized by FDA) lists leukocyte reduction as an alternative to CMV seronegative products.

Filtration technology for leukocyte reduction has greatly improved since publication of the original study. Today all red cell units are leukocyte reduced by filtration and platelets are leukocyte reduced by apheresis. Many transfusion services consider leukocyte reduced products to be CMV safe and automatically substitute them for CMV negative orders in adult patients.

Each unit of red blood cells contains about 250 mg of iron complexed with hemoglobin, which is 100 times more than the quantity absorbed in the daily diet. Since the body has no mechanism for excretion of excess iron, iron overload can occur after as few as 10 transfusions. Transfusion hemosiderosis may become apparent after about 100 units of blood. Iron overload causes oxidative damage to the liver, heart, pancreas, thyroid, and other endocrine glands. Organ damage may already be advanced at the time of diagnosis. Organ toxicity begins when reticuloendothelial sites of iron storage become saturated and iron becomes deposited in other cells.

The most serious complication is cardiotoxicity, which may lead to arrhythmias, congestive heart failure and death. Hepatic injury, diabetes mellitus and adrenal insufficiency may also occur.

Serial ferritin monitoring is helpful in assessing total body iron burden. Treatment with iron chelation agents, such as parenteral deferoxamine, should be initiated early in the course of chronic transfusion therapy. Deferasirox is an oral iron chelator that can aid in management of iron overload. Long term maintenance of serum ferritin below 300 ng/mL is associated with improved survival.

Adverse effects of this drug include GI distress, auditory and ocular disturbances, hepatic and renal failure, and bone marrow suppression. The EPIC study demonstrated reduction in iron burden and a clinically acceptable safety profile in patients with sickle cell anemia (Br J of Haematol. 2011; 154; 387-397)

The best way to prevent iron overload is to limit the number of transfusions as much as possible. Multiple studies have demonstrated an improvement in iron overload after transition from simple transfusion to erythrocytapheresis (Transfus Med Hemother.2008; 35: 24-30; J Pediatr Hematol / Oncol. 1996; 18: 46-50; Blood. 1994; 83: 1136-1142). However, erythrocytapheresis is more expensive and may be associated with additional complications.

Each unit of red blood cells contains about 250 mg of iron complexed with hemoglobin, which is 100 times more than the quantity absorbed in the daily diet. Since the body has no mechanism for excretion of excess iron, iron overload can occur after as few as 10 transfusions. Transfusion hemosiderosis may become apparent after about 100 units of blood. Iron overload causes oxidative damage to the liver, heart, pancreas, thyroid, and other endocrine glands. Organ damage may already be advanced at the time of diagnosis. Organ toxicity begins when reticuloendothelial sites of iron storage become saturated and iron becomes deposited in other cells.

The most serious complication is cardiotoxicity, which may lead to arrhythmias, congestive heart failure and death. Hepatic injury, diabetes mellitus and adrenal insufficiency may also occur.

Serial ferritin monitoring is helpful in assessing total body iron burden. Treatment with iron chelation agents, such as parenteral deferoxamine, should be initiated early in the course of chronic transfusion therapy. Deferasirox is an oral iron chelator that can aid in management of iron overload. Long term maintenance of serum ferritin below 300 ng/mL is associated with improved survival.

Adverse effects of this drug include GI distress, auditory and ocular disturbances, hepatic and renal failure, and bone marrow suppression. The EPIC study demonstrated reduction in iron burden and a clinically acceptable safety profile in patients with sickle cell anemia (Br J of Haematol. 2011; 154; 387-397)

The best way to prevent iron overload is to limit the number of transfusions as much as possible. Multiple studies have demonstrated an improvement in iron overload after transition from simple transfusion to erythrocytapheresis (Transfus Med Hemother.2008; 35: 24-30; J Pediatr Hematol / Oncol. 1996; 18: 46-50; Blood. 1994; 83: 1136-1142). However, erythrocytapheresis is more expensive and may be associated with additional complications.

Recombinant tissue plasminogen activator (rtPA) has been approved to treat ischemic stroke in the first three hours following the onset of symptoms. If given promptly, 1 in 3 patients who receive rtPA have major improvement in their stroke symptoms.

Recombinant tPA produces local thrombolysis by converting plasminogen into plasmin, which then degrades fibrin into fibrin split products. More than 50% of rtPA is cleared 5 minutes after cessation of the infusion and approximately 80% is cleared after 10 minutes. Despite this rapid clearance, rtPA prolongs the prothrombin and activated partial thromboplastin times and decreases fibrinogen levels for as long as 24 hours or more from the time of infusion.

An important complication after treatment of acute stroke with rtPA is symptomatic intracerebral hemorrhage (sICH). Studies suggest that between 3.5% and 6% of stroke patients treated with rtPA develop sICH, and the hemorrhagic complication leads to death in about 50% of patients. Older patients, patients with very severe strokes, and patients who used aspirin before their strokes are all at increased risk for sICH (Yaghi S et al. Symptomatic intracerebral hemorrhage in acute ischemic stroke after thrombolysis with intravenous recombinant tissue plasminogen activator. JAMA Neurol 2014; DOI: 10.1001/jamaneurol.2014.1210).

Because coagulopathy related to rtPA lasts up to 24 hours, both early and sustained reversal is necessary to avoid hematoma expansion and neurologic deterioration. The American Heart Association/American Stroke Association treatment guidelines for symptomatic ICH management recommend replacement of coagulation factors and platelets, acknowledging that there is limited evidence to support the strategy. Specifically, the guidelines call for the use of 10 bags of cryoprecipitate to reverse coagulopathy and one bag of single donor platelets or 6 to 8 bags of random donor platelets.

Each bag of cryoprecipitate contains 200 to 250 mg of fibrinogen and will increase the plasma fibrinogen level of a 70-kg adult by 6 to 8 mg/dL. Generally, 10 bags of cryoprecipitate are given if the fibrinogen level is between 50 and100 mg/dL and 20 bags are given if it is less than 50 mg/dL. A fibrinogen level should be measured at 30 to 60 minutes after completion of the transfusion to determine if additional doses are needed. The therapeutic goal is to keep the plasma fibrinogen level above 100 mg/dL. Circulating half life of fibrinogen is 3 to 5 days.

The potential benefits of aggressive approaches to managing sICH must be weighed against the potential for worsening thrombosis.

Vel is a high-prevalence RBC antigen that was discovered in 1952. Only 1 in 4000 people are Vel negative. In addition, some people have very weak expression of Vel antigen by serological testing.

The SMIM1 gene codes for the Vel antigen. The Vel negative phenotype is caused by a homozygous 17 base pair deletion in exon 3 of the SMIM1 gene which leads to a frame shift mutation and premature stop codon. No Vel protein is transcribed. The Vel^weak phenotype is due to a heterozygous deletion in the same exon, resulting one functional copy of the SMIM1 gene.

If a patient who is Vel negative is transfused with Vel positive red blood cells, they may form anti-Vel alloantibodies. These antibodies can be clinically significant. Some antibodies do not cause a hemolytic transfusion reaction, while others cause severe reactions. Likewise, Vel antibodies can cause mild to severe hemolytic disease of the fetus and newborn (HDFN).

Vel is a high-prevalence RBC antigen that was discovered in 1952. Only 1 in 4000 people are Vel negative. In addition, some people have very weak expression of Vel antigen by serological testing.

The SMIM1 gene codes for the Vel antigen. The Vel negative phenotype is caused by a homozygous 17 base pair deletion in exon 3 of the SMIM1 gene which leads to a frame shift mutation and premature stop codon. No Vel protein is transcribed. The Vel^weak phenotype is due to a heterozygous deletion in the same exon, resulting one functional copy of the SMIM1 gene.

If a patient who is Vel negative is transfused with Vel positive red blood cells, they may form anti-Vel alloantibodies. These antibodies can be clinically significant. Some antibodies do not cause a hemolytic transfusion reaction, while others cause severe reactions. Likewise, Vel antibodies can cause mild to severe hemolytic disease of the fetus and newborn (HDFN).

Most patients with autoimmune hemolytic anemia (AIHA) have a positive direct antiglobulin test (DAT) for IgG, complement or both. However, approximately 10% of patients presenting clinically with AIHA have a negative DAT. Physicians need laboratory evidence of an immune basis for hemolytic anemia before starting immunosuppressive therapy.

There are at least three potential causes of a negative DAT in a patient with AIHA:

• RBC bound IgG below the threshold of detection of the routine DAT
• Low affinity IgG that washes off RBCs during the washing phase of the tube test DAT
• RBC bound IgA or IgM that is not detected by routine antiglobulin reagents

The routine tube DAT has a lower limit of detection of about 200 IgG molecules per red blood cell. A more sensitive method is needed to detect cases associated with lower levels of autoantibody. The most commonly available methods include: enzyme linked antiglobulin test (ELAT), flow cytometry, direct Polybrene test, solid phase and column agglutination.

Low affinity IgG can sometimes be retained on red blood cells by washing them with ice cold saline or low ionic strength saline (LISS) and then retesting using routine anti-IgG and anti-C3d reagents. Column agglutination tests may also detect low affinity IgG autoantibody because red cells are not washed prior to testing.

IgM autoantibodies usually fix complement, which can be detected by anti-C3d reagent. Anti-IgM and anti-IgA antisera are available commercially but are not approved for clinical use by the FDA in the United States. Approximately 2% of AIHA cases are caused by IgA autoantibodies.

Many of these more esoteric tests are not available in hospital transfusion services. Testing can be sent to reference laboratories that are often located within regional blood centers.

The G antigen is present on almost all red blood cells expressing the Rh D or C antigens. It is absent from red cells that are D and C negative. Rh negative patients can produce anti-G antibody following transfusion of Rh negative, C antigen positive red blood cells. Anti-G is a clinically significant antibody that can cause hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. Anti-G is usually considered less likely to cause severe hemolytic disease than ant-D or anti-C.

When an antibody panel is performed to identify an unexpected antibody, anti-G appears as a combination of anti-D and anti-C antibodies. Rh negative patients with an anti-G antibody can be safely transfused with red blood cells that are negative for both the D and C antigens. Therefore, additional adsorption studies are not necessary to distinguish anti-G from anti-D plus anti-C.

The only situation in which anti-G should be definitively identified is when a D negative woman of child bearing age is pregnant and is a candidate for  Rh immune globulin. Anti-G may mask the presence of anti-D on standard antibody panels. Subsequent adsorption and elution studies are needed to distinguish anti-G from anti-D. If anti-G is detected, then Rh immune globulin should be given. If anti-D is present, the patient is not a candidate for Rh immune globulin.

Red blood cells with the r” (cE) phenotype are the least likely to express the G antigen because they lack both D and C antigens.

Shirey RS, et al. Differentiation of anti-D, -C and –G: Clinical relevance in alloimmunized pregnancies. Transfusion 1997;37:493-496. 

Von Willebrand Factor (vWF) plays a key role in both platelet plug and fibrin clot formation. When endothelial damage occurs, vWF multimers bind to exposed subendothelial collagen and to platelets, promoting platelet adhesion and aggregation at the site of injury. Activated platelets initiate the coagulation cascade resulting in the formation of a fibrin clot.

During the past 55 years, many publications have documented the association of aortic stenosis and bleeding from gastrointestinal angiodysplasia. Bleeding associated with aortic stenosis was found to be caused by excessive proteolysis of high molecular weight vWF multimers under conditions of increased shear stress. Aortic valve replacement usually cures GI bleeding due to replenishment of the largest vWF multimers within a few hours.

Bleeding due to defects in vWF structure or function that are not inherited, but are consequences of other medical disorders, has been classified as acquired von Willebrand syndrome (AVWS) to distinguish it from congenital von Willebrand disease (vWD). More recently, excessive bleeding associated with other cardiovascular disorders such as ventricular septal defect, hypertrophic obstructive cardiomyopathy, and placement of left ventricular assist device (LVAD) has also been attributed to development of AVWS.

Development of AvWS and mucosal bleeding may be an additional indication for consideration of surgical correction of the underlying cardiovascular disorder. Laboratory detection of AVWS may provide an additional tool to evaluate the efficacy of surgical management. Unfortunately, the panel of tests recommended to diagnose congenital vWD (vWF antigen, vWF activity and FVIIIc) are usually normal in AVWS. Laboratory confirmation of AVWS requires VWF multimer analysis to detect the loss of high molecular weight multimers. This analysis involves a labor-intensive assay involving separation of vWF multimers by protein electrophoresis and detection of all molecular weight forms by Western Blot. Multimer analysis is only available at a few reference laboratories and has a long turnaround time. Results may not be available in time for clinical decision making.

Another sensitive indicator of impaired vWF function in cardiovascular disorders with high shear stress is the whole blood platelet function screen performed on the PFA-100 analyzer. This assay is very sensitive for detection of vWD and AVWS. Prolonged closure times with both COL/EPI and COL/ADP are typical of either vWD or AVWS. One 5.0 mL sodium citrate (light blue top) tube is required. Sample must be received by the laboratory within 3 hours of collection.

Various transfusion therapies have been tried to treat excessive bleeding associated with these cardiovascular disorders including plasma, desmopressin (DDAVP), aprotinin,  tranexamic acid, aminocaproic acid and recombinant FVIIa (Novoseven). However, none of these products specifically addresses the specific underlying problem. Replacement of loss of high molecular weight vWF can best be achieved by transfusion of cryoprecipitate or a factor concentrate that contains Factor VIII and vWF, such as Humate P.