You order a complete blood count (CBC), but the hematology lab reports back that the patient’s blood sample cannot be read by the automated hematology analyzer. They suggest you resend blood and keep the sample at 37 degrees F during transport to the lab. You make arrangements to send such a sample, and the CBC is successfully completed (next slide).

For more information on how cold agglutinins interfere with the CBC, click here.

The following is the original complete blood count (CBC) on blood at room temperature (1) and warmed blood (2):

UNABLE TO RE, unable to report

Let’s reorganize the CBC on the warmed blood sample according to our trusted TBP format:

WBC (109/L)Hb (g/dL)Hct (%)MCV (fL)RDW-SD (fL)PLT (109/L)

What’s what: WBC, white blood cell count; Hb, hemoglobin; MCV, mean cell volume; MCHC, mean cellular hemoglobin concentration; RDW-SD, red cell distribution width-standard deviation; platelets, PLT; Normal values: WBC 5-10 x 109/L, RBC 4-6 x 1012/L, Hb 12-16 g/dL, Hct 35-47%, MCV 80-100 fL, MCHC 32-36 g/dL, RDW-SD < 45 fL, platelets (PLT) 150-450 x 109/L

A question for the more experienced:

For more information on how cold agglutinins interfere with the CBC, click here.

Differential diagnosis of normocytic anemia. Note that the first step is to check the reticulocyte count.

The reticulocyte count was 5%. Is this an appropriate reticulocyte count in the setting of anemia? (we may want to change the number to higher)

Read more here

Another way to determine whether the reticulocyte count is appropriate or not is to consider the absolute reticulocyte count. This is provided by some labs. In other cases, it can be calculated from the red cell count and the % reticulocytes (maybe include in the link out to retics on previous slide).

For example:

Reticulocyte %Red blood cell countAbsolute reticulocyte count
1%5 x 1012/L50 x 109/L
5%5 x 1012/L250 x 109/L
10%5 x 1012/L500 x 109/L

An absolute reticulocyte count of > 120 x is considered an appropriate response to anemia!

So, the patient appears on the “appropriate reticulocyte count” side of the diagnostic ledger:

He shows no signs of bleeding, so you decide to focus on the possibility of hemolytic anemia.

Sort the lab parameters according to their expected blood levels in hemolysis?

Indirect bilirubin
Plasma hemoglobin
Direct bilirubin

A few hours later, his hemolysis labs return [please provide normal ranges, we can then make a table]:

  • AST 25
  • ALT 30
  • ALP 170
  • LDH 300
  • Tbili 2.5 [indirect should be high!]
  • Dbili 2.0
  • Haptoglobin <8

True or false: the mean cell volume (MCV) is always normal in a patient with elevated reticulocytes.

Because reticulocytes are larger than mature red blood cells, their presence in increased numbers may increase the MCV.

Thus far, the data support a diagnosis of hemolytic anemia. Before we consider the peripheral smear, let’s discuss an approach to hemolysis.

What is hemolytic anemia?

In normal physiology, red cells last approximately 120 days in circulation. As they circulate, they gradually develop wear-and-tear damage as they pass through small capillaries or across cardiac valves, as well as oxidative damage to their membranes. Eventually, this damage makes them less flexible. They become unable to navigate the narrow splenic sinuses, and are eventually phagocytosed by splenic macrophages.

“Hemolysis” is an umbrella term that refers to the premature destruction of red blood cells – they are destroyed before reaching the point of physiologic, senescent death. There are a wide variety of processes that can lead to hemolysis, which we will explore below.

What can cause hemolysis?

 Key point: there are a wide variety of causes. It helps to have a systematic approach.

What are some approaches for generating a differential diagnosis?

1. Mnemonic-associated approaches can be useful, especially for broad differentials. A popular mnemonic is shown below:


Mnemonics can help ensure you maintain a broad differential, without missing a key group of etiologies. However, mnemonics are nonspecific. Sometimes it can be helpful to consider a physiologic or anatomic approach to formulating your differential.

2. A diagnostic schema offers a systematic approach to generating a differential diagnosis for a particular problem. Schemas generally subdivide your differential into various physiologic “buckets”. We will explore a diagnostic schema for hemolysis on the next slide.

Causes of hemolysis. First question is whether it is immune or non-immune. A negative direct antiglobulin test (DAT) virtually rules out immune causes. On the non-immune side, we can divide causes into those that are intrinsic to the red blood cell (the red cell is “at fault”) and those that are extrinsic to the red blood cells (arising from the cell’s environment, the otherwise normal red cells suffering collateral damage). Extrinsic causes include thrombotic microangiopathy (TMA, which includes disseminated intravascular coagulation, thrombotic thrombocytopenia purpura and hemolytic uremic syndrome), microangiopathic hemolytic anemia (through abnormal heart valves, or from marathon running or Congo drum playing), infection (for example, malaria, babesiosis and clostridium perfringens), spur cell anemia (and the related condition, Zieve syndrome), burns and copper excess. The intrinsic causes include hemoglobinopathies (thalassemia and sickle cell disease), membrane defects (hereditary spherocytosis, heredity elliptocytosis, paroxysmal nocturnal hemoglobinuria), and enzyme defects (G6PD deficiency, pyruvate kinase deficiency).

This schematic places the differential diagnosis of hemolytic anemia into the wider context of anemia.

Let’s organize the differential diagnosis of hemolytic anemia in table format and consider appropriate next steps, including a peripheral smear, blood work and imaging:

Smear (in addition to polychromatophilia)Other testsComments
Immune-mediated hemolysisSpherocytes, agglutinationDirect antiglobulin testThis cannot be ruled out in our patient
TMASchistocytes, thrombocytopeniaRenal function, ADAMTS13Typically presents with acute illness and low platelet count
Spur cell anemiaAcanthocytes, target cellsLiver function tests, liver imagingNo evidence of liver disease
InfectionSmear positive for parasitesBlood cultures (clostridium)No recent travel, no known exposure to ticks
MAHASchistocytesValve imagingNo recent valve repair or replacement, not a marathon runner, does not play drums with hands
Sickle cell diseaseSickle cells, nucleated RBCs, Howell-Jolly bodiesHb electrophoresisNot the right demographics
ThalassemiaMicrocytic red blood cells, target cellsHb electrophoresis, DNA sequencingCannot rule out
Hereditary spherocytosisSpherocytesOsmotic fragility test/EMA binding testCannot rule out. Patients can present in adulthood
Heredity elliptocytosisElliptocytesOsmotic gradient ektacytometry Cannot rule out. Patients can present in adulthood
Paroxysmal nocturnal hemoglobinuriaOften normalFlow cytometryCannot rule out
G6PD deficiencyBite cell during acute hemolytic episodeG6PD activity levelsCannot rule out. Patients can present in adulthood
PK deficiencyNucleated red cells, acanthocyte-like cellsPK activity levelsShould have presented earlier in life

Note from the table about how helpful the peripheral smear is in differentiating causes of hemolysis! Almost every cause of hemolysis has unique findings on the smear.

Match the smear (A-C)) with the condition:

Correct! Sorry, Incorrect.

Match the smear (D-F) with the condition:

Spur cell anemia
Correct! Sorry, Incorrect.

The patient’s smear performed on warmed blood is similar to the following (want to show smear with clumping at room temperature?):

Further classifying hemolytic anemias

In our diagnostic schema for hemolysis, we discussed one method to subdivide hemolysis – separating etiologies into immune and non-immune buckets, and non-immune bucket into “extrinsic”, and “intrinsic” causes. This is helpful for developing a broad differential for hemolysis. However, there are other ways to classify hemolysis; one common method is to separate these into “intravascular” and “extravascular” causes.

Extravascular hemolysis occurs within the spleen and reticuloendothelial system (RES), in a similar manner to the physiologic destruction of senescent red cells reviewed earlier in this case. However, this destruction occurs too early, often due to intrinsic defects within the red cell. For example, hereditary spherocytosis results in extravascular hemolysis – the abnormal shape and reduced flexibility of the spherocytes results in the cells getting “caught” in splenic sinusoids. As a result, they are treated the same way as aging red cells – they are phagocytosed by splenic macrophages.

On the other hand, intravascular hemolysis occurs by an entirely different mechanism. Rather than the phagocytosis described above, hemolysis occurs inside the blood vessel. For example, microangiopathic hemolytic anemias result in intravascular hemolysis. Due to the formation of microthrombi in capillaries and other small vessels, red cells are sheared apart as they try to navigate these vessels.

This distinction matters because the location of hemolysis (intravascular or extravascular) can help characterize the ways in which hemolysis will manifest.

Sort the cause of hemolysis with the predominant mechanism:

Hereditary spherocytosis
Spur cell anemia
Sickle cell disease
G6PD deficiency
Extravascular hemolysis
Intravascular hemolysis

Key point: the “RBC-killer” (i.e. clot/shearing, complement/MAC, infectious organism, oxidative stress) has to be inside the blood vessel in order for the hemolysis to occur in the intravascular space.

Key point: spleen/RES-hemolysis is facilitated by mononuclear phagocytes (monocytes/macrophages) looking for structurally-abnormal or antibody-coated RBCs. This phagocytosis can result in spherocytes.

The ability of hemolytic labs to differentiate between intravascular or extravascular hemolysis is by no means perfect. At first glance, we might assume that intravascular causes of hemolysis lead to elevated leakage products including LDH, AST and plasma hemoglobin (released into the circulation by lysed cells), while extravascular causes of hemolysis lead primarily to increased indirect bilirubin (from intracellular conversion of Hb to bilirubin).


  • Many cause of hemolysis are associated with both intravascular or extravascular hemolysis
  • Both intravascular or extravascular hemolysis may be associated with a similar biochemical profile:
    • Intravascular hemolysis causes release of:
      • LDH
      • AST
      • Hemoglobin (Hb)
        • Free Hb binds haptoglobin (Hp) leading to reduced levels of Hp.
        • Hb-Hp complexes are taken up by macrophages, converted to indirect bilirubin.
    • Extravascular hemolysis leads to:
      • Macrophage engulfment of red blood cells.
      • Intracellular degradation of red cell Hb into bilirubin.
      • Leakage of LDH and AST back to the circulation.

For an in-depth explanation of Coombs testing, consider this 5-minute video.

Your patient’s results:

Coombs (DAT) positive for complement protein C3d (4+) and negative for IgG.

Cold-agglutinin screening positive. Patients may be screened for presence of cold antibodies if DAT positive for complement and/or IgG by repeating DAT at room temperature or by other methods approved by individual institutions. The result of a cold agglutinin test is typically reported as a titer, such as 1:64 or 1:512, expressed as the inverse value of the highest serum dilution at which agglutination can be detected, or the thermal amplitude. Some clinicians feel that cold agglutinin titer evaluation and thermal amplitude tests may not be necessary for many cases and can be used as supportive evidence for diagnosis.

Which of the following statement(s) is/are correct?

Cold agglutinins are autoantibodies that bind to their antigen at an optimum temperature of 3–4°C.
DAT positive for complement with or without immunoglobulin (Ig) G
IgG reported in 21%-28% of cases.
Clinically significant cold agglutinins should be differentiated from normally occurring cold agglutinins
Low-titer cold agglutinins are present in the plasma of all adults. They are polyclonal. low titer anti-I (usually < 1:64 at 4 degrees C [39.2 degrees F]; rarely > 1:256); low thermal amplitude (usually < 25 degrees C [77 degrees F]).
Cold agglutinins usually target the ABO blood group system
They target the Ii antigen system. Levels of I and i antigens on red blood cell (RBC) surface are inversely proportional; neonatal RBCs express mostly i antigen; after 18 months of age, I antigen predominates; therefore, anti-I is more pathogenic than anti-i in children and adults

Our patient was found to have antibodies directed against I antigen.

The Ii antigen system is a human blood group system:

  • The letter “I” was introduced in 1956 to denote the high degree of “individuality” shown by a patient whose RBCs lacked the antigen.
  • Based upon a gene on chromosome 6 (IGnT gene).
  • Consists of the I antigen and the i antigen.
  • I and i antigens are carbohydrate structures on membrane glycoproteins and glycolipids.
    • i antigen
      • The predominant structure on fetal RBCs.
      • Consists of a straight-chain polymer of at least two lactosamines.
      • After birth, an enzyme becomes activated that can add additional lactosamine disaccharides to galactose, resulting in a branched chain structure that constitutes the big I antigen.
    • I antigen
      • Present on RBCs in the vast majority of the population (approximately 99 percent).
  • The I/i antigens on RBCs exhibit a reciprocal expression profile during development: the I antigen is normally present on the cell membrane of red blood cells in all adults, while the i antigen is present in fetuses and newborns. In newborn infants the i antigen undergoes gradual conversion to reach adult levels of the I antigen within 18 months of birth. 
  • The function of the big I and little i antigens is unknown.

Cold agglutinins should not be confused with cryoglobulins. Cryoglobulins are immunoglobulins (with or without complement protein) that precipitate in vitro at temperatures below 37°C and redissolve on rewarming. They are not associated with hemolysis. Cryoglobulinemia syndrome should be suspected in patients presenting with:

  • Arthralgia
  • Purpura
  • Skin ulcers
  • Glomerulonephritis
  • Peripheral neuropathy

A closer look at the mechanism of hemolysis in cold agglutinin disease is shown on the following slide.

In cold agglutinin disease, pentameric IgM autoantibodies, usually IgM (kappa subtype), are produced against the I antigen. They bind to its antigen at the erythrocyte surface during passage through the cooler parts of the peripheral circulation, causing 1) agglutination of red blood cells impairing navigation of small peripheral capillaries and leading to ischemic symptoms which are most prominent in the fingers and toes, and 2) binding of components of the classical pathway of complement, such as C1, C4, and C2. C1 esterase then activates C2 and C4, generating C3 convertase which cleaves C3 into C3a and C3b. Upon warming to 37°C in the central circulation, cold agglutinins detach from the cells, allowing agglutinated erythrocytes to separate, while C3b remains bound. C3b-opsonized cells are prone to phagocytosis by the mononuclear phagocytic system, mainly in the liver, a process known as extravascular hemolysis. On the surface of the surviving erythrocytes, C3b is cleaved, leaving high numbers of C3d molecules which can be detected by the DAT. Because there are no C3d receptors in the mononuclear phagocyte system, these erythrocytes are protected from phagocytosis. In some patients and situations, however, C3 is supposed to initiate the terminal complement cascade by binding and splitting C5, resulting in the formation of the C5b-9 complex (membrane attack complex) and intravascular hemolysis.

There are two types of cold agglutinin disease:

  • Primary (idiopathic) cold agglutinin disease (CAD):
    • > 90% of patients reported to have clonal expansion of kappa-positive B cells in bone marrow and a monoclonal IgM-kappa paraprotein.
    • Monoclonal cold autoantibody is often encoded by IGHV4-34 and targets I antigen.
  • Secondary CAD (referred to by some as cold agglutinin syndrome):
    • Arises in the setting of an underlying disorder such as
      • Viral infection
      • Autoimmune disorder
        • Systemic lupus erythematosus (SLE)
        • Rheumatoid arthritis
      • Overt lymphoid malignancy
    • Antibody is typically IgM and its target depends on underlying condition:
      • Mycoplasma pneumonia – polyclonal IgM against I antigen
      • Epstein-Barr virus – polyclonal IgM against i antigen
      • Cytomegalovirus – polyclonal IgM against i antigen
      • Aggressive non-Hodgkin lymphoma (NHL) – monoclonal IgM against I antigen, but light chain restriction can be lambda as well as kappa

Younger patients may be more likely to have an underlying infection or autoimmune disorder, and older individuals (eg, >60 years of age) may be more likely to have a lymphoid (B-cell or plasma cell) malignancy such as aggressive non-Hodgkin lymphoma or Waldenström macroglobulinemia (WM).

Of note, infectious and inflammatory causes tend to result in a temporary surge in polyclonal antibody production, and therefore tends to produce a less severe clinical picture.

In contrast, lymphoproliferative disorders tend to result in monoclonal anti-I production, which can result in a more significant degree of anemia and symptom burden.

Source: Clinical Hematology International 2(3) 95–100

It is important to understand why your patient is producing anti-I antibodies. Is this a “primary” disorder (i.e. CAD), or is there another underlying process (i.e. CAS)? Because CAS can be associated with lymphoid malignancy, it is particularly important to evaluate for underlying causes. What tests would you order to distinguish primary from secondary cold agglutinin disease? (We can place here or in subsequent visit)

Serum electrophoresis and immunofixation
Bone marrow examination
It is important to consider evaluation for lymphoproliferative disorders in a high-risk patient (eg. in a patient who is older, has a more chronic anemia, or has features to suggest malignancy).
Testing for HIV, hepatitis B, and hepatitis C
Testing for an infectious disorder is appropriate in those with fever and/or respiratory symptoms, particularly if the duration of symptoms is short.
Anti-double-stranded DNA and antinuclear antibody testing to help diagnose autoimmune disorders
Especially if patient has arthralgias or arthritis, malar rash, or cytopenias

These tests were ordered in our patient and showed: XXXX

Remember our patient presented with acrocyanosis and livedo reticularis. Are these findings consistent with cold agglutinin disease?

Acrocyanosis and/or Raynaud’s phenomenon reported in 40%-90% of cases. Livedo reticularis is also common.

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