Sample Lesson – Coombs (Indirect vs. Direct), Hemolytic Anemia, and What a “Type and Screen” Actually Does

 

 

Next Step: Make Cards on the Automatic Key Concepts, and Vignettes

Remember, the more you automatically know what each sentence means on your test, the better you will do. There are 4 stages in making interpretation more automatic:

  • Stage 1: Unable to Make Pathophysiologic Chronologies in Either Timed or Untimed setting
  • Stage 2: Basic Pathophysiologic Chronologies, but with Significant Gaps
  • Stage 3: Detailed Pathophysiologic Chronology Without Time, but Unable to Consistently Generate PC During Timed Setting
  • Stage 4: Consistent Pathophysiologic Chronologies in Timed Setting

My goal with these vignettes is to help you reach Stage 4. How do you do so?

  • With the Automatic Key Concept cards, you can master the underlying information to move past Stages 1 + 2.
  • Then, with the Vignette/Pathophysiologic Chronology cards, you can teach yourself to make these connections on your exam.

LFT Summary:

 

 

summary of key concepts:

 

 

Automatic Key Concepts:

Copy + paste these into your cards, to make these key concepts more automatic.

 

B cell receptor – what is it? How does it affect the function of a B cell?

Immunoglobulin (antibody) plus CD79. When the B cell is activated, the B cell receptor is what will eventually become the antibody produced by the plasma cell

 

T cell-dependent vs. T cell-independent B cell activation: what are the advantages of using T-cells?

T-cell-dependent allows for:

    • Affinity maturation (somatic hypermutation)
    • Isotype switching
    • Memory B cell formation

 

What kinds of molecules (protein, carbohydrates, fat) can activate T cells? Why would this make sense?

Protein. MHC presents antigens to T cells, and presents protein antigens only. Thus, T cells are primarily only activated against protein antigens

 

If I wanted a vaccine to promote IgG antibodies against a carbohydrate antigen, could I use only the carbohydrate antigen itself in the vaccine? Why?

No. Unlike B cells, T cells cannot be activated against carbohydrate antigens. No T cell activation → no T-cell dependent B cell activation → no isotype switching (and no memory B cells/affinity maturation)

 

Conjugate vaccine – what is it? Why would you do this?

A vaccine where the antigen you want to make antibodies against is conjugated (linked) to a protein antigen. This ensures that T cells can be activated against the antigen → T cell-dependent B cell activation.

This is a common strategy for making vaccines against non-protein antigens (e.g., vaccines against encapsulated organisms like the Hib, PCV13 pneumococcal, N. meningiditis vaccines)

 

Conjugate vs. unconjugated polysaccharide vaccine – what isotypes would you expect? Why?

Conjugate – can produce ANY isotypes (usually IgG, though)

Because of the protein conjugate, can activate T cells → T cell dependent B cell activation → isotype switching → can produce other isotypes

Unconjugated – can ONLY produce IgM

Because no protein conjugate, CANNOT activate T cells → T cell independent B cell activation → no isotype switching → IgM only produced

 

 

Affinity maturation – explain the process to describe why it benefits a B cell

Allows for the B cell receptor to bind with greater affinity for its target antigen. It does this by creating many B cells, each with a slightly different version of the B cell receptor. The B cells with the best BCR survive, and the others die (apoptose).

 

 

T cell-dependent vs. T cell-independent B cell activation: which produces higher-affinity antibodies? Why?

T-cell dependent B cell activation → affinity maturation → clonal B cell proliferation with better-binding B-cell receptors surviving → ultimately, higher-affinity antibodies for the antigen

 

What antibody isotype is best for protection of mucosal surfaces?

IgA

 

T cell-dependent vs. T cell-independent B cell activation: which would be better for an infection against bacterial pneumonia?

T-cell dependent B cell activation → isotype switching → allow production of IgA antibodies → protection of mucosal surfaces → better protection against encapsulated bacteria

 

Hemagglutinin – use its name to explain what it causes in vitro

A protein on influenza that causes agglutination (clumping) of RBCs – hence the name heme (RBC) agglutinin (agglutination)

It binds to the sialic acid residues on respiratory epithelium (and incidentally RBCs) → internalization of virus into respiratory epithelial cells

NOTE: there is no clear pathological significance to the fact that influenza binds to RBCs, although it is used in the clinical hemagglutination assay, which quantifies the relative amount of pathogen/antibody

 

Antibodies – do they bind better at warm or cold temperatures? Why?

Virtually all things (including antibodies) bind better at colder temperatures. This is because there is less thermal (kinetic) energy forcing the molecules apart

 

Warm vs. cold agglutinins – what are they, actually?

Warm agglutinin – antibody that can cause agglutination at warm AND cold temperatures

Cold agglutinin – antibody that can cause agglutination at cold temperatures ONLY

Technically, ALL antibodies bind better in the cold

 

Warm vs. cold agglutinins – use the meaning behind each name to explain what antibody isotype you would expect them to be

Often warm agglutinins are IgG antibodies. Warm agglutinins cause agglutination (bind well) at both warm (where binding is harder) AND cold temperatures (where binding is easier). This is usually because they have undergone affinity maturation from T cell dependent B cell activation, thus allowing them to do isotype switching as well.

Often cold agglutinins are IgM antibodies. Cold agglutinins cause agglutination at cold temperatures ONLY (where binding is easier due to the lower thermal energy). This is because they have NOT undergone affinity maturation from T cell dependent B cell activation, indicating they have also NOT undergone isotype switching

 

Direct Coombs Test – if you see agglutination vs. do not see agglutination, what does that mean?

See agglutination → patient’s RBCs had pre-existing antibodies bound to them PRIOR to adding the Coombs reagent (antihuman antibodies)

No agglutination → patient’s RBCs did NOT have pre-existing antibodies bound to them prior to adding the Coombs reagent

Key point: Direct Coombs looks at whether RBCs are ALREADY coated with antibody

 

What are the steps in the indirect Coombs test?

Take SERUM from the patient → add RBCs with known antigens on them → add anti-human globulin → look for whether they agglutinate or not

 

Indirect Coombs test – when would you expect to see agglutination?

If the patient has anti-RBC antibodies (e.g., anti-Rh antibodies) in their serum, and the RBCs have the antigen (e.g., they are Rh+) → agglutination

In other words, indirect Coombs is positive if the patient has atypical antibodies

 

Direct vs. indirect Coombs tests – use what you take from the patient to explain what they are looking for

 

Direct Coombs – “Did immune-mediated hemolysis happen?”

Take RBCs from the patient; you are looking for whether the patient’s RBCs have antibody on them already (e.g., you are assessing whether the patient had a hemolytic transfusion reaction – a positive Direct Coombs’ test is the “smoking gun”)

Indirect Coombs – “Are there antibodies that could cause immune-mediated hemolysis?”

Take SERUM (and any antibodies) from the patient; you are looking for whether the patient has antibodies in their blood that would reject certain RBCs (e.g., you are doing an antibody screen to see whether the patient can receive blood containing certain minor antigens like Kidd, Kell, or Duffy)

 

Direct vs. Indirect Coombs test – when would you perform these?

Direct Coombs – “Did immune-mediated hemolysis happen?”

If you suspect immune-mediated hemolysis (e.g., work up a suspected hemolytic transfusion reaction)

Vs.

Indirect Coombs – “Are there antibodies that could cause immune-mediated hemolysis?”

To assess for antibodies against RBCs, usually prior to transfusion (the “screen” in a “type and screen”) or in pregnancy (it’s the test for anti-Rh antibodies for Rh-negative women)

 

 

 

labs interpretation:

 

Explain the relationship between the Direct Coombs test, hemoglobin, and bilirubin. Which bilirubin is most prominent?

Direct Coombs positive = RBCs coated with antibodies. In this context, it indicates autoimmune hemolytic anemia → RBC destruction → hemoglobin ↓ / production of bilirubin ↑ → indirect bilirubin >> direct bilirubin

vignette/pathophysiologic chronologies:

A 28-year-old woman comes to the physician with 4 weeks of worsening fatigue and dyspnea. She is otherwise healthy. Her temperature is 37ºC, pulse is 98/min, blood pressure is 115/75 mmHg, and respirations are 18/min. On exam, she has pallor, mild jaundice, and splenomegaly. Significant labs are shown.

What is the pathophysiologic chronology?

Summary:

AIHA → anemia + direct Coombs positive → HR/CO ↑ to compensate for lower O2 content of blood

Detailed:

Born healthy → (unclear etiology) immune activation against protein antigen on own RBCs → T-cell dependent B cell activation → affinity maturation + isotype switching → IgG ”warm” agglutinins → AIHA (autoimmune hemolytic anemia) → immunoglobulin-coated RBCs cleared by spleen →

    • Back-up of blood in spleen/splenic work hyperplasia → splenomegaly
    • Anemia (pallor) → oxygen carrying capacity ↓ (= 1.34 * hemoglobin * O2 sat + PaO2 * 0.003) → HR ↑ → CO ↑ → maintain O2 delivery to tissues
    • RBCs already coated by antibodies → Direct Coombs positive (RBCs clump when add anti-human globulin)
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