Immunology Lab

Question 1

What is a cryoglobulin?

Answer 1

By the original definition, they are antibodies that precipitate from plasma at 4 degrees Celsius and re-dissolve when reheated to 37 degrees Celsius.

Ultimately, this is due to the antibody’s affinity for antigen, but there are three subtypes of cryoglobulins.

Question 2

Cryoglobulinemia type I

Answer 2

Monoclonal cryoglobulins

May be IgM, IgG, IgA, or Bence-Jones protein (free light chains)

This is often seen in patients with hematologic neoplasms and can cause hyperviscosity syndrome.

This type of cryoglobulinemia tends to present as Waldenstrom’s or Multiple myeloma rather than as a classical cryoglobulinemic vasculitis.

Question 3

Cryoglobulinemia type II

Answer 3

Monoclonal and polyclonal immunoglobulins

A type of mixed cryoglobulinemia. The monoclonal component may be IgM, IgG, or IgA, but the polyclonal component is usually IgG.

In essence, the monoclonal protein is often RF and the other immunoglobulins are along for the ride. Like type III cryoglobulineia, the antigen of the monoclonal component is typically another immunoglobulin.

This tends to present as a classic cryoglobulinemic vasculitis and may be associated with Hep C, RA, CLL, or as essential cryoglobulinemia. Strongly associated with Hep C.

Question 4

Cryoglobulinemia type III

Answer 4

Polyclonal cryoglobulins (usually IgM) that is bound to polyclonal antibodies (IgM, IgG, IgA, IgE)

Can contain RF or multiple RFs, but less classical for RF than type II. Like type II cryoglobulineia, the antigen of a portion of the monoclonal component is typically another immunoglobulin.

Present as a vasculitis and may occur in SLE, essential cryoglobulinemia, many viral infections (HBV, HCV, HIV, EBV, CMV), biliary cirrhosis, or rarely with bacterial infections.

Question 5

What percentage of people with mixed cryoglobulinemia have Hep C?

Answer 5

~92%

Question 6

Ways of reporting cryoglobulin precipitation

Answer 6

1) Cryocrit (% of total volume)
2) Protein content
3) Ig content

Question 7

Immunodiffusion

Answer 7

Question 8

Reaction of identity

Answer 8

Question 9

Reaction of non-identity

Answer 9

Question 10

Reaction of partial identity

Answer 10

Question 11

SPEP peaks

Answer 11

Question 12

Main parameters for a clinical diagnosis of cryoglobulinemic vasculitis

Answer 12

No specific guidelines, but generally:

1. Clinical syndrome of purpura, arthralgias/arthritis, weakness
2. Serum mixed cryoglobulins with RF activity
3. Low C4
4. Leukocytoclastic vasculitis on biopsy

Question 13

Why is mixed cryoglobulinemia associated with Hep C?

Answer 13

The exact reason is unclear, but it is thought that HCV-infected lymphocytes are under the influence of NS5A.

HCV-E2 has also been suggested as a possibility, as some reports suggest it can activate VDJ recombination in antigen-exposed B cells.

Many of the affected circulating cells also demonstrate t(14:18).

Question 14

The vast majority of cold agglutinins are against. . .

Answer 14

. . . the i-antigen or I-antigen (same as the EBV and Mycoplasma pneumoniae-induced antibodies, respectively)

Sometimes the epitope recognized by a cold agglutinin may include both the I/i variant and the ABO variant antigens: For example, anti-IA, anti-IB.

Question 15

I-antigen and i-antigen

Answer 15

Ubiquitous cell membrane carbohydrate antigens

i is predominates in fetal cells. When an individual matures, an enzyme becomes activated which constitutively modifies i to I. So, most children over age 2 and adults have predominantly I antigen.

However, in ~1% of the population, this enzyme is lacking, and so i predominates. In adults, we see approximately 99% I phenotype and 1% i phenotype.

Question 16

Kappa and Lambda in serum vs urine

Answer 16

Serum: Lambda chain present as a dimer. K/L ratio ~0.5 (0.26-1.65), with about twice as much lambda as kappa despite that twice as much kappa is made as is labmda. This is due to the slow renal clearance of lambda. In patients with advanced renal disease, the ratio flips to 1.2, as clearance in these patients is primarily by the reticuloendotheilal system which is not affected by size.

Urine: Lambda chain dissociated as monomers.

pH 8.6 gel: Lambda chain dissociated as monomers.

Question 17

Where different classes of Ig are found on SPEP

Answer 17

Beta 1: Haptoglobin, thrombin, transferrin – no Igs

Beta 2: C3, C4, IgA

Between Beta 2 and Gamma: IgM

Gamma: IgG

Question 18

Meltzer’s triad

Answer 18

The triad of cryoglobulinemic vasculitis symptoms:

1. Palpable purpura
2. Arthralgias/Arthritis
3. Weakness

Question 19

Pseudothrombocytosis

Answer 19

When immune complexes (often cryoglobulin-mediated) are read as platelets due to their size

Can correct for this by re-warming the sample and agitating prior to repeating CBC.

Question 20

Serum vs plasma

Answer 20

Plasma is everything noncellular

Serum is what you get once you let platelets and fibrin deplete by clotting.

Note – you have to be careful with serum for cryoglobulin mesaurement, as cryoglobulin precipitants may become nonspecifically trapped within the clot.

Question 21

5 “types” of hyperlipidemia

Answer 21

Type 1: Clear tube with creamy layer on top. Chylomicrons present at the surface – due to chylomicron load overwhelming LPL activity.

Type 2: Clear tube. Cholesterol in the form of LDL. Patient will have HTN, HLD, PVD.

Type 3: Clear tube. Cholesterol in the form of VLDL. Patient will have HTN, HLD, PVD, and xanthomas.

Type 4: Cloudy tube. Triglycerides. Due to relative insulin deficiency.

Type 5: Cloudy tube with creamy layer on top. Mix of at least type 1 and 4 with chylomicrons and triglycerides.

Note that none of these have a precipitant – so any precipitation should still be suspicious for cryoproteins after fibrin is ruled out.

Question 22

Cryoglobulin-associated hemolysis

Answer 22

Mostly classical complement mediated.

Usually due to a low-titer IgM, but can also be due to high-titer IgG.

C3 convertase is activated and C3b is deposited on the RBC membrane. CD55 and CD59 present on the RBC membrane prevent intravascular hemolysis by interfering with the late complement pathway (except in cases of extreme inflammation and rapid complement activation – this is paroxysmal cold hematuria).

CR2 (aka CD21) on reticuloendothelial MΦ mediate C3b uptake at the spleen, resulting in extravascular hemolysis.

In the lab, IgM often has diffused away, but red cells will remain coated in C3b, which we can detect. If IgG was the cause, IgG may be present on red cells with or without C3b.

Question 23

Why do we never see an IgE cold agglutinin?

Answer 23

IgE is a poor precipitator

Remember – IgE is primarily a receptor adaptor for FcεRI and 99% of blood IgE is cell-bound. It did not evolve to precipitate antigen the way IgG, IgM, and IgA did.

Very rare case reports have been described.

Question 24

Serum free light chain

Answer 24

Measured using antibodies against the “hidden” kappa and lambda epitopes

Has prognostic value in almost every stage of plasma cell dyscrasia, including “nonsecretory” myelomas and risk of progression of MGUS to myeloma.

Question 25

Cryoglobulin vs Cold agglutinin

Answer 25

Cryoglobulin: anti-antibody

Cold agglutinin: anti-red cell membrane