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


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.


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.


Blood Components 101

This will serve as a guide for the specific indications, storage requirements and stability of the different blood components.


Whole Blood, Packed Red Blood Cells: 1-6 degrees Celsius.

Plasma, Cyroprecipitated AHF: -18 degrees Celsius.

Platelets: 20-24 degrees Celsius with continuous gentle agitation.

Granulocytes: 20-24 degrees Celsius without agitation.


Whole Blood: When refrigerated a unit of whole blood has a shelf life between 21-35 days depending on the additive that is used. Must be transfused within 4 hours when at room temperature.

Packed Red Cells: Packed red cells are stable for up to 42 days refrigerated, but they can also be frozen with glycerol as a cyroprotectant for up to 10 years. They must be deglycerolized by washing and thawed prior to transfusion and must be transfused within 24 hours once thawed.

Platelets: Platelets have a shelf live of only 5 days. Some hospitals and clinics are extending the shelf life out to 7 days with continuous bacterial testing to ensure there is no contamination.

Plasma: Plasma products must be processed and frozen within 8 hours of collection and are stable for 12 months. Once thawed they must be transfused within 24 hours.

Thawed Plasma: Has an expiration of 5 days.

Cryo: Cyro AHF once pooled and frozen has a stability of up to 12 months.

Granulocytes: Granulocytes must be transfused within 24 hours after donation.



Whole Blood: Used to replace the loss of both RBC mass and plasma volume. The product is 550-600 mL of whole blood, with a hematocrit of about 40%.

Packed Red cells: Usually the red cell product of choice. 330 mL of red cells, hematocrit of about 55-65% with an additive solution.



Platelets: Platelets derived from whole blood must contain at least 5.5×10^10 platelets in 40-70 mL of plasma in at least 90% of the units tested. Platelets donated through apheresis must contain at least 3×10^11/L platelets in 100-500 mL of plasma. One apheresis platelet collection is equivalent to six pooled random donor platelet concentrates.


Asset 13b-Plasma resizeda

Plasma: Can be derived from whole blood or apheresis collection. Plasma contains albumin, coagulation factors, fibrinolytic proteins, and immunoglobulins. Fresh frozen plasma (FFP) derived from whole blood is usually 220-300 mL and units derived from apheresis usually contain 400-600 mL. The plasma must be frozen within 8 hours of collection.



Cryoprecipitated Antihemophilic Factor (AHF): AHF is prepared from FFP. It is slowly thawed, then refrozen within one hour of thawing. AHF typically contains 5-20 mL of plasma with 80-120 U/concentrate of Factor VIII, 150-250 mg/concentrate of fibrinogen, 40-70% of vWF, and 20-30% of Factor XIII that would normally be present in FFP. Making it the treatment of choice for Von Willebrands Disease and Hemophiliacs.


Red Cells

Red cell transfusions are used to treat hemorrhage and to improve oxygen delivery to tissues. The decision to transfuse red cells should be based on the patients clinical condition. Indications for red cell transfusion include acute sickle cell crisis, acute blood loss of greater than 30% of blood volume, or patients with symptomatic anemia that can’t function without red cell repleting. The threshold for transfusion of red cells should be a hemoglobin of 7 g/dL in adults and children. Maintenance can be at a level of >7-9 g/dL.  One unit of red cells should increase the hemoglobin by 1 g/dL and hematocrit by 3%.

Washed Red Cells

Washed red cells are washed with saline to remove any residual plasma proteins. These are used for patients with a history of allergic transfusion reactions. These patients have an IgA deficiency and have developed anti-IgA.

Leukocyte Reduced

Leukocyte reduced red cells decrease the incidence of febrile transfusion reactions. They are indicated for those at high risk of transfusion-associated GVHD or transfusion-related immune suppression. For a unit to be considered leukocyte reduced, there must be less than 5×10^6 leukocytes.

Irradiated Red Cells

Used for patients with a history of febrile transfusion reactions or patients that are immunocompromised immediately after an allogeneic bone marrow or stem cell transplant. Patients at risk for HLA-GVHD will receive irradiated red cells.


Plasma transfusion are recommended for patients with active bleeding and an international normalized ratio (INR) greater than 1.6. Its indicated for patients on anticoagulant therapy that are undergoing an invasive procedure. Plasma should not be administered for a high INR without active bleeding. Plasma is indicated for patients with inherited clotting factor deficiencies for which there is no safe recombinant factor available. Those factors are II, V, X, and XI. Plasma is used as an emergent reversal of warfarin (coumadin) toxicity to prevent intracranial hemorrhage. It is also used in acute disseminated intravascular coagulation (DIC) or other thrombotic microangiopathies such as thrombotic thrombocytopenic purpura (TTP) or hemolytic uremic syndrome (HUS). Plasma is often times transfused with red cells during massive transfusions; with the definition of massive transfusion being greater than 5,000 mL in an adult of average weight (70 kg).


Platelet transfusions are indicated to prevent hemorrhage in patients with thrombocytopenia or those with functional platelet defects. Contradictions for platelet therapy are patients with TTP and heparin-induced thrombocytopenia (HIT) as transfusion in these clinical situations can result in exacerbation of thrombosis. Platelet transfusions can be used prophylactically in invasive surgeries with no active bleeding and commonly used in active bleeding situations along with transfusion of FFP and red cells. One unit of apheresis platelets should increase the platelet count in adults by 30-60×10^9/L.

Transfusion of neonates is complicated and should be based on upon clinical reasons with consideration to the platelet count. If the count is <20×10^3/mL, you should always transfuse if possible. When you reach 20-30×10^3/mL you should consider transfusion, but weigh all possibilities. In a case of active bleeding, transfusion is absolutely appropriate, but all factors should be considered. Transfusion is also indicated in there is signs of a coagulation disorder, intraventricular or intraparenchymal cerebral hemorrhage, an invasive procedure, or if there is alloimmune neonatal thrombocytopenia.

Cryoprecipitate AHF

Cryo contains high concentrations of factor VIII and fibrinogen and is used especially in cases of hypofibrinogenemia. Hypofibrinogenemia is typically seen in the setting of massive hemorrhage or in a consumptive coagulopathy such as DIC. Indications for cyroprecipitate AHF are factor VIII and factor XIII deficiency, congenital fibrinogen deficiency, and von Willebrand disease.



Granulocyte Pheresis

Indicated for patients with fever, neutropenia, septicemia or an antibiotic resistant bacterial infection.

















Blood System Portfolio: ABO Group

The ABO blood group system was first discovered in 1901 by Karl Landsteiner. In 1902 Sturli and Von Decastello discovered the AB group. The ABH antigens do not develop until about 6 weeks of fetal life and the concentration of antigens increases until 3-4 years of age and level out. Antibodies to the ABO blood group are naturally occurring, meaning that the body will develop immunity without previous exposure to the other blood type antigens. They are high titer antibodies and will bind an activate complement in vivo. Antibodies are not able to be detected in serum until 3-6 months of age and titers decrease markedly after the age of 65.

When classifying an individuals blood type is it typically referred to by the forward and reverse groupings. For example, if someone is blood type A, they will have naturally occurring antibodies to type B. That is why it is very important when selecting blood products for a patient that every precaution is taken to match the blood types as best as possible. The ABO antibodies are clinically significant and the hemolytic reactions are immediate and hemolytic.


The ABH genes that encode for the ABO blood group system encode for a glycosyl transferase enzyme that adds a specific monosaccharide to a precursor substance which results in a distinguishable antigenic structure. The B gene encodes for a-3-D-galactosyltransferase which adds the D-galactose sugar resulting in the B antigen on the RBC surface. The A gene encodes for a-3-N-acetylgalactosaminyltransferase which adds the N-acetyl-D-galactosamine sugar resulting in the A antigen. The H gene encodes for the a-2-L-fucosyltransferase which adds the L-fucose sugar resulting in the H antigen.

The ABH antigens are membrane bound glycolipids. All ABH antigens have type 2 precursors that are chains linked by a 1,4 linkage. To have an A antigen, the H antigen MUST be present. Same with the B antigen, but absence of the A and B antigens with the H antigen present is considered type “O”. The ABO genes are located on chromosome #9 and there are 4 major alleles; A1, A2, B and O. A1 is codominant with B. A2 is recessive to A1, but also codominant with B. O is recessive to all other alleles. The H gene is located on chromosome #19 and there are 2 alleles that can be expressed. H encodes for fucosyltransferase, and “h” is an amorph. Individuals that have the phenotype “hh” are considered to have the bombay phenotype and are extremely rare. These individuals will not produce the H antigen, therefore there are no A or B antigens as well. It doesn’t matter if the genes for the A and B antigens are present, without the H antigen, there can be no A or B antigen production.

ABO antibodies are primarily IgM that react at room temperature and sometimes at 37 degrees celsius. The antibodies follow Landsteiners law which states an individual possesses antibodies to ABO antigens that are absent from their own cells. As mentioned above, the forward and reverse grouping should agree. If there is a discrepancy there is further testing that can be done to distinguish the problem. Discrepancies can be found in a previous article written titled “ABO discrepancies”. One such test is called the anti-A1 lectin test. 1-8% of A2 individuals will develop anti-A1, although not clinically significant, it can cause discrepancies. Lectin is a plant seed extract that agglutinates specific human cells. It agglutinates to A1 cells, but not A2 cells. Its possible to use this test to help resolve discrepancies.

Stay tuned for the next blood group system discussed.


Blood Draw Tube Colors and Order

The tube order may not seem like a big deal and may seem unnecessary to some, but it is very important to pay attention too. It also matters as to what type of needle is being used for the draw. If a butterfly needle is being used it is important to have a spit tube because with a butterfly there is air within the hose that connects the needle to the vacutainer. Its important to get this air out before filling any tubes used for patient testing. If a standard needle is being used, you typically don’t need the spit tube, but its good practice. The order still remains the same for each.

Light Blue: The typical tube for routine coagulation studies. The additive is sodium citrate (3.2% or 3.8%). Citrate is a anticoagulant which binds to calcium within the blood so the blood can’t clot. Calcium plays an important role in primary and secondary homeostasis. See my post on DIC for that information, in short it is used in the coagulation cascade. An important aspect of coagulation studies is that the light blue tube must, must be filled completely. There is a ratio of sodium citrate to whole blood and that must remain constant. The tube must be rejected if it is not filled completely.

Green or Mint Tubes: These tubes are used for chemistry studies. Often referred to as PST or plasma separator tubes. The additive in these tubes are sodium heparin, lithium heparin or ammonia heparin. The heparin, being an anticoagulant activates antithrombin, which blocks the coagulation cascade and produces a whole blood with plasma sample instead of a clotted blood and serum sample. When these tubes are centrifuged, the gel barrier moves upwards creating a barrier that separates the plasma from the red cells allowing the plasma to be aspirated directly for testing.

Gray Tube: The gray tube tops are typically used for glucose testing, ethanol levels or lactate level testing. The additive is potassium oxalate and sodium fluoride. Potassium oxalate is an anticoagulant which prevents clotting and the sodium fluoride is an anti-glycolytic which prevent the cells from using the glucose in the sample.

Lavender/Pink Tube: The lavender tube is typically used for hematological testing or for Hemoglobin A1C testing. The pink tube is used primarily for blood bank testing such as type and screen and cross-matching. The additives in the lavender and pink tubes are EDTA K2 or EDTA K3. The EDTA binds to calcium which blocks the coagulation cascade in the same way that citrate in the light blue tube does. Red cells, leukocytes, and platelets are in EDTA anticoagulated blood for 24 hours. Blood smears should be done within 3 hours of receiving the sample.

SST/Mustard Tube: Serum separator and clot activator that will separate the blood from the serum upon centrifugation. This tube is usually used to test for aldosterone, B12, ferritin and folate levels.

There are not all the different tubes that are used, but these are the most common tubes that I listed and the ones that a laboratory professional will most likely come across. Its important to understand the additive in each one to make sure that they are appropriate for the testing that needs to be done on the patient sample itself within the tube.

phb_Order of Blood Draw with labels72dpi