Understanding SPEP/Immunofixation,UPEP and free light chains

Basic understanding of Monoclonal gamapathy

Normal plasma cells secrete antibodies directed against specific antigens. It is capable of producing 5 different heavy chains (IgA, IgG, IgM,IgD,IgE)and 2 different light chains(Kappa and lambda). So, there is a possible 10 different combination of antibodies(IgG kappa, IgG lambda, IgM kappa, IgM lambda and such)For some reason there is always an excess of light chains than heavy chain which in physiologic state is excreted into the tubules but reabsorbed  completely and broken down into amino acids (recycled by tubules). If there is an excess of light chains than what the tubules can handle(as in the case of monoclonal gamapathy), light chains are excreted in urine (Benze jones protein) .

In Monoclonal gamapathy(MM, Waldenstroms and such) there is clonal proliferation of plasma cell that produces the same antibodies/clonal(not directed against antigen). Some clonal plasma cells produce just light chains and not heavy chains. Rarely some clonal plasma cells produce heavy chains and not light chains.

Now let us see what information we could get from SPEP/Immunofixation, UPEP and free light chains.


Method : Serum proteins are electrophoretically separated in SPEP based on its electrical charge. Serum proteins generally categorize in 5 different zones( albumin, alpha 1 globulin, alpha 2 globulin, beta 1 globulin, beta 2 globulin and gamma) Gamma region has the polyclonal immunoglobulin. If there is a monoclonal protein , we see a spike in the gamma region(occasionally the M spike can be seen in the alpha or beta region). The peak of the M spike in electrophoresis in relation to the total protein will help quantify the M protein. say if the spike is 40% of the total protein , then if we know the total protein quantification , we should be able to calculate the amount of M protein ex / total protein in 10 gm/dl and spike is 40% , then M protein is 4 gram/dl . So, SPEP helps determine two information.

1) The presence of M protein

2) Quantification of M protein


This is used in conjunction with SPEP. By using antibodies against the 5 different known classes of heavy chain and 2 different classes of light chain, we can determine the type of M protein. So, the immunofixation helps

1) Identify the type of M protein such as IgG Kappa or IgM lambda and such..



Some light chains are freely filtered in the glomerulus and quickly cleared from blood. So, SPEP cannot identify these light chains.Under these circumstances, UPEP will increase the sensitivity of identifying the myeloma protein when combined with SPEP. SPEP alone has 80% sensitivity and combining UPEP increases the specificity to 95%. The UPEP is done the same way as SPEP and can quantify the M protein just as in SPEP.

Free light chains

Technically, if we can obtain serum free light chains , we do not need to order UPEP since SPEP and free light chains can together increase sensitivity of identifying M protein to >95%. It is just that it is expensive compared to SPEP and UPEP combined together.


In short,

SPEP — For knowing if there is M protein and if so quantification of M protein

Immunofixation – For identifying the M protein (using antibodies agains heavy and light chains)

UPEP – For light chains in urine

Free light chain — Highly sensitive for identifying even small increase in M protein-expensive though!








Glomerulonephritis – diagnosis based on immune deposit


40 y/o with PMH significant for hep C -untreated and recent history of sore throat presents with photosensitivity rash and generalized weakness. Evaluation shows AKI, anemia, hypercalcemia and SPEP with M spike.UA shows dysmorphic RBC. Serologies are pending !

What could be the etiology of the suspected GN !

a) Hep C related MPGN

b) Post streptococcal GN

c)  Lupus nephritis

d) proliferative GN related to Monoclonal gammapathy

e) Cryoglobulinemic GN related to HepC, Lupus or Monoclonal gammapathy.

This imaginary case brings to focus the diagnosis of GN based on immune deposit.

Of course, the clinical presentation and serologies will indicate the possible diagnosis in real clinical situation! The case presented above and the following explanation is purely for understanding the immune deposits based classification of GN (Which is very useful in teasing out the etiology of GN)


1) Light microscopy in all possible diagnosis may show proliferative GN

2) Immunofluorescence may show

a)  Immune complex mediated disease process( by which I mean immunoglobulin deposition +/_ c3 deposition

If IgA dominant  ———–> IgA nephropathy or IgA dominant post infectious GN( Staph super antigen and such)

If IgG dominant and monoclonal (Either kappa or lambda and not both)——-> Monoclonal gammapathy associated proliferative GN

If IgG dominant and polyclonal (both kappa and lambda)——–> Post infectious GN

If IgM dominant and polyclonal ——————————> Chronic infection such as hepatitis and autoimmune disease

If IgA, IgG, IgM (all Ig classes, C3 and C4- full house or partly full house in appropriate clinical setting)——-> Lupus nephritis and other auto immune diseases

b) No or negligible immune deposit ; pauci-immune GN ——————> ANCA + or ANCA – vasculitis

c) Complement mediated (by which I mean trace or minimal Ig deposit)

C3 dominant with minimal immunoglobulin of any subtype  C3 GN or DDD

This understanding of immunofluorescence finding coupled with location of immune deposit in EM and complement levels will aid the diagnosis

C3 low and C4 normal—-> post infectious GN or shunt nephritis( typically staph epidermidis in patients with chronic shunt)

C4 low and C3 normal or low normal —-> Cryoglobulinemic GN from any cause(Polyclonal gammapathy, Hep C or auto immune)

Both C3 and C4 low —-> lupus nephritis

Normal complements—> ANCA + or ANCA – pauci-immune vasculitis, Good pasture disease(anti-GBM mediated), IgA nephritis or Infective endocarditis related GN which most commonly presents as pauci-immune GN.

Positive cryoglobulin in blood may or may not be pathogenic unless demonstrated as deposits in glomerular capillary loop!


Hope this helps in understanding the immune deposits in GN and its role in identifying the etiology of proliferative GN in a complex case as the one mentioned above.