DARA-T Workup

Daratumumab (Darzalex) is an IgG1k monoclonal antibody directed against CD38, which is over expressed on the plasma cells in patients with multiple myeloma. Daratumumab binds to CD38 and causes apoptosis through antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity. In 2015 the FDA approved daratumumab for the treatment of refractory multiple myeloma. Refractory meaning that patients have received at least three previous treatment protocols that failed to show sustained efficacy or any efficacy at all. Recently in May of 2018, the FDA approved daratumumab for first line therapy in combination with bortezomid, melphalan, and prednisone. The names of the drugs aren’t important, what is important is that this monoclonal antibody approach has become more common and now has moved into first line therapy meaning that more patients are going to receive this treatment. Its no secret that patients with multiple myeloma when undergoing treatment and throughout the course of the disease progression need blood component transfusions.

Typing and screening patients that are receiving daratumumab is extremely difficult and time consuming. The daratumumab not only binds to the CD38 on the malignant lymphoma cells, but it also binds to the red cells who express CD38. This causes interference in transfusion testing. Part of normal pre-transfusion testing is an antibody screen. An antibody screen is important as it tells the transfusion team if there are any alloantibodies. Alloantibodies are antibodie directed towards red cell antigens on the donor cells. If a patient has an alloantibody, it makes selecting red cells for transfusion difficult. Additional testing must be done to select antigen negative donor cells for the antibody that the recipient or the patient has. Daratumumab causes the antibody screen and corresponding antibody panel panreactive, including a positive autocontrol. This may mask any additional clinically significant alloantibody that the patient may have.

The blood bank team must perform testing prior to the patient receiving this daratumumab. The clinical team must be in communication with the blood bank. Before the patient receives the medication, the team must get a baseline type and screen. Normally they are negative, but in the off chance that they have an alloantibody, the blood bank can identify the antibody before daratumumab interferes with testing. Other testing must include a complete phenotype of the patients cell. A complete phenotype will identify all the antigens that are present on the patients cells. This tells the blood bank and clinician vital information. If the patient does NOT have the antigen present on their red cells, there is a chance that they can produce an antibody towards that antigen on donor cells making it hard to find correct donors for transfusion. For example, if the patient is negative for the E antigen, they may or may not develop an antibody towards the E antigen, so in the event that the donor red cells have the E antigen present, the patients antibody will attack those cells and cause a transfusion reaction. For the characteristics of different transfusion reactions, reference transfusion reactions.

Once the daratumumab has been given there are techniques that must be followed to obtain a sample that is suitable for testing. An enzyme called dithiothreitol (DTT) is used to negate the binding of DARA-T to CD38 on the red cell surface. This will allow for an antibody screen to be run. Unfortunately, DTT destroys the Kell antigen on the red cell surface. Kell is a clinically significant antibody in transfusions so its important to know whether or not if the patient has the antigen or not. Patients treated with DTT, MUST have Kell negative donor units, because of the risk of developing an anti-K antibody and not being able to identify it.

 

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The Pseudohyponatremia Debacle

Measurement of electrolytes in a sample of plasma is probably one of the most common tests that is performed. Every patient who gets blood drawn will also get a Chem 7 or CP13 which will include an analysis of electrolytes. There are two different technological approaches to measuring electrolytes, depending on what kind of chemistry analyzer is employed. Some use an indirect ISE and some are centered on direct ISE.

When a whole blood sample is used it is typically centrifuged beforehand to separate the sample into its plasma and red cell layers. Measurement of the electrolytes will involve using the plasma portion of the sample. Plasma consists of 93% water and approximately 7% of solid constituents, mainly proteins and lipids. The electrolytes being measured are somewhere in the water portion the plasma.

The two different methods; indirect and direct ISE differ in that direct ISE is able to respond to the electrolyte concentration within the plasma water while the indirect measures the electrolyte concentration by volume of total plasma. Total plasma including the 93% water content and 7% protein and lipid portion. That is an important distinction because the distribution of the water and protein content of plasma is important. The ratio of water/protein content is not always going to be 93/7. Depending on the patient that ratio can be skewed and inaccurate results can be reported.

So lets recap, the direct ISE measures the electrolytes using the water content of plasma, while the indirect ISE measures the electrolytes using the ratio of water/protein in whole plasma.

Indirect ISE

Indirect ISE measures electrolytes using a total plasma sample that has been diluted with diluent. This requires that the whole blood sample has been centrifuged and separated. The method measures the mean concentration in the entire plasma; water and protein content of plasma. The concentration is then multiplied by the dilution factor.

Pseudohyponatremia

Variation of the content of proteins and lipids from a normal with cause an error in the reported electrolyte results when using an indirect ISE. In particular, when measuring sodium (Na+). Dilution, as needed in the indirect ISE involves taking a pre-determined volume of the “total” sample, not only the water content of the plasma, and adding to a diluent. The total number of measurable ions in the sample is expressed as the average concentration in the total volume of the original sample, because of this the reported values depend on the protein and lipid content of the samples. A patient with hyperlipidemia will skew the 93/7 ratio which will cause falsely decreased levels of electrolytes, most often sodium. If the low sodium level is due to lipids, its possible to centrifuge to clarify the sample to get a more accurate result. Sometime the best thing to do is to rerun the sample using direct ISE. If its possible to get a whole blood sample then use point-of-care analyzers, like an i-STAT, or some blood gas analyzers (ABG) which can give a more accurate result.

Direct ISE

Direct ISE measures electrolytes using non-diluted whole blood or a plasma sample. The actual measurement that is gained is based on the water content of the plasma. It measures the electrolyte activity in the plasma water. The electrochemical activity of the ions in the water is converted to the readout concentration by a fixed multiplier that is ion-specific. The use of the fixed factor reflects the actual, clinically significant result, irrespective of the level of proteins or lipids within the solid phase of plasma.

 

 

Serum Protein Electrophoresis (SPE)

Protein electrophoresis measures the specific proteins in the body by using electrical charge to separate them. Serum proteins are either albumin or they are globulins. Globulins are then further delegated into either gamma globulins or alpha-1, alpha-2, or beta globulins.

The normal ranges for each protein fraction in the serum is as follows;

Albumin: 3.6-5.2 g/dL

Alpha-1: 0.1-0.4 g/dL

Alpha-2: 0.4-1.0 g/dL

Beta: 0.5-1.2 g/dL

Gamma: 0.6-1.6 g/dL

Normal SPE

SPE is useful in the diagnosis of Multiple Myeloma or Waldenstrom macroglobulinemia which presents as a characteristic elevation in the gamma globulin peaks. It can also be used to help diagnose liver diseases, renal diseases, anemia, and even malnutrition.

Based upon which serum protein fraction is either decreased or increased gives evidence to a specific diseases or a group of diseases.

Albumin is produced by the liver and is the most abundant and arguably one of the most important proteins in the body. One of its main functions is to maintain the colloid pressure between the tissues and the bloodstream. Increases in albumin are seen in severe dehydration. They are decreased in malnutrition, liver disease, nephrotic syndrome or severe burns.

The major alpha-1 globulin is alpha-1 antitrypsin, which is produced in the lungs and in the liver. Increases in alpha-1 are seen in inflammatory states and pregnancy. Alpha-1 is decreased in alpha-1 antitrypsin deficiency. Alpha-1 antitrypsin deficiency is used as marker for an increased risk of emphysema.

Alpha-2 globulins include serum haptoglobin, alpha-2-macroglobulin, and ceruloplasmin. Haptoglobin binds to any free hemoglobin in the blood as a result of intravascular hemolysis to prevent its excretion by the kidneys. Ceruloplasmin is the major protein in the body that carries copper which also plays a role in iron metabolism. Increases in alpha-2 are also seen in inflammatory states and nephrotic syndromes. In cases of nephrotic syndromes you will see a decrease in albumin and a compensatory increase in alpha-2. Alpha-2 may also be elevated in hyperthyroidism, steroid use, and oral contraceptives. It is usually decreased in hemolysis and liver disease.

Beta-globulins include transferrin, low-density lipoproteins (LDL) and complement proteins. Transferrin is the molecule that is used to transport dietary iron to the liver, spleen and bone marrow for storage. LDL is the major carrier of cholesterol in the blood. Complement is a branch of the immune system that plays a specific role in the inflammatory response. Beta protein fractions are increased in hyperlipidemia and iron deficiency anemia. It is decreased in malnutrition.

Gamma-globulins encompass the different classes of immunoglobulins. Gamma globulins are increased in either monoclonal or polyclonal gammopathies. It is decreased in agammaglobulinemia and hypogammaglobulinemia. Immunoelectrophoresis is a reflex test that detects the levels of the different immunoglobulins within the gamma-globulin which should be used when there is an abnormal amount of protein detected.

serum electrophoresis with molecules included

A monoclonal gammopathy is a narrow band increase in the gamma protein fraction that is composed of a single class of immunoglobulins secreted by a malignant clone of plasma cells. It is also known as the M-protein. M-protein is typically representative of a diagnosis of multiple myeloma, but can be detected in other lymphoid malignancies. Its important to understand that absence of the M-protein does not rule out monoclonal gammopathies as sometimes there is not a detectable concentration within the serum. Sometimes this can lead to a false-negative result. More sensitive tests such as a serum immunofixation test should be performed. Multiple myeloma is a monoclonal increase in IgG immunoglobulins. Other monoclonal gammopathies include waldenstrom macroglobulinemia which is an increase in IgM immunoglobulins. Some more common ones are Al amyloidosis and monoclonal gammopathy of undetermined significance (MGUS).

Polyclonal gammopathies are infectious or various inflammatory processes characterized by a broad-based peak in the gamma fraction. This typically represents a polyclonal immunoglobulin increase seen in autoimmune disease, liver diseases, viral/bacterial infections, and various other malignancies.