Test of the Month

Laboratory testing for cryoglobulins

Gabriela Motyckova,1,2 and Mandakolathur Murali1,2,3*
Cryoglobulins are immunoglobulins that precipitate below 378C and can cause multiorgan damage. There
are three types of cryoglobulins: Type I (also called simple), which is mostly associated with monoclonal
gammopathy and/or other hematologic disorders and Type II and Type III (known as mixed cryoglobulins),
which are associated with infectious and systemic diseases. Testing for cryoglobulins is complicated by
lack of reference range, standards, and stringency in maintaining testing temperature conditions. Identifica-
tion of cryoprecipitate can be critical for patient care; therefore, correct testing conditions are crucial for
reliable cryoglobulin testing. The patient’s blood sample should be kept at 378C initially to avoid premature
precipitation of cryoglobulins and thereby decreasing the yield for subsequent identification. This could
cause a false negative result. After warm centrifugation or warm cell precipitation, the clear serum is
observed at 48C for formation of cryoprecipitate. The cryoprecipitate is then washed in cold buffer, and the
resulting precipitate is warmed to 378C and subjected to further analysis by immunodiffusion and immuno-
fixation. In addition to Meltzer’s triad of purpura, weakness and arthralgias, cryoglobulinemias have protean
manifestations involving skin, joints, kidney, nervous system, as well as the hematopoietic system. The
management of cryoglobulinemia especially in patients with organ damage remains difficult. Treatment of
cryoglobulinemia focuses on management of the underlying lymphoproliferative disorder or infectious or
systemic causes. Medical management may also include corticosteroids and other immunosuppressive
agents and plasmapheresis. Rituximab therapy seems to abrogate the aberrant B cell response. Am. J.
Hematol. 86:500–502, 2011. V C 2011 Wiley-Liss, Inc.

Introduction clinical and laboratory features of the cryoglobulinemia

Cryoglobulins are immunoglobulins which precipitate syndromes.
below 378C from patient’s serum and redissolve when Laboratory Testing for Cryoglobulins
rewarmed in vitro [1,2]. They were first observed in 1933 For cryoglobulin identification, two 10 mL red top tubes
by Wintrobe and Buell [3] in a patient with Raynaud’s phe- of sample (without an anticoagulant) are drawn from a
nomenon. They were called cryoglobulins (cold precipitable patient and transported to the laboratory at 378C (com-
serum globulin) in 1947 [4]. In the 1960s, a clinical profile monly, this is achieved by submerging the tubes in warm
of palpable purpura, asthenia, arthralgia, and renal disease water during transportation to the laboratory). The sample
was recognized, caused by immune complex deposition is allowed to clot at 378C for 1 hr. The clot is separated
and cryoglobulinemia [5]. There are three types of cryoglo- from the sides of the tube with a Pasteur pipette, and the
bulins depending on their characteristics and interactions serum is separated by warm (378C) centrifugation and ali-
with other proteins (Table I) [6]. quoted into three tubes at 378C. One tube is the Wintrobe
The laboratory workup of suspected cryoglobulin should tube for quantification of the cryoprecipitate (a Wintrobe
be performed in patients with unexplained multiorgan dis- tube has markings that are used to quantify). The second
ease involvement such as skin, liver, renal, peripheral nerve tube contains a larger quantity of serum, which is used for
manifestations, Raynaud’s syndrome, and in patients with cryoprecipitate observation and subsequent analysis. The
the typical triad (first described by Meltzer and hence third tube is used to determine the solubility of the cryopre-
known as Meltzer’s triad) of purpura, weakness, and cipitate on rewarming to 378C, a feature of cryoglobulin. If
arthralgias [5]. a warm centrifuge is not available, serum should be sepa-
Monoclonal cryoglobulinemia (Type I) is often seen in rated in a 378C water bath after allowing cells to clot and
patients with hematologic malignancies and can cause settle at 378C for an hour without centrifugation, being
hyperviscosity syndrome. Mixed cryoglobulinemia (Types II careful to ensure that the red cells and fibrin clot are not
and III) often presents as a systemic vasculitis due to aspirated with the serum. It is important for the sample
immune complex deposition in the small vessels and may temperature not to drop below 378C until serum separation
be the presenting feature in patients with infectious and is complete to avoid premature precipitation. Improper sam-
systemic disorders [7]. Patients may present with palpable ple handling may result in a missed diagnosis of cryoglobu-
purpura, weakness, arthralgia as well as liver and renal linemia [10]. Increasing concentration of cryoglobulins
involvement, peripheral neuropathy, sicca syndrome, Ray-
naud’s phenomenon, and B cell non-Hodgkin lymphoma
[1]. Around 92% of patients (range 70–100% depending on Conflict of interest: Nothing to report.
1
Massachusetts General Hospital Cancer Center, Boston, Massachusetts;
patient population) with mixed cryoglobulinemia have Hepa- 2
Dana-Farber Cancer Institute, Boston, Massachusetts; 3Department of
titis C infection (HCV) versus 1.8% having Hepatitis B [8,9]. Medicine, Massachusetts General Hospital, Boston, Massachusetts
Conversely, about 40–66% of patients with HCV have ele-
*Correspondence to: Mandakolathur Murali, Clinical Immunology Laboratory,
vated circulating monoclonal and/or polyclonal immunoglo- Massachusetts General Hospital, 55 Fruit Street, Boston, Massachusetts
bulins. The clinical syndrome of cryoglobulinemia is mani- 02114. E-mail: [email protected]
fested in only about 5% of HCV patients [7]. Among the Received for publication 11 February 2011; Revised 23 February 2011;
Type III cryoglobulins, the mixed cryoglobulinemia can be Accepted 26 February 2011
associated with organ involvement and small and medium Am. J. Hematol. 86:500–502, 2011.
size vessel vasculitis and is again seen in infectious and Published online 14 March 2011 in Wiley Online Library (wileyonlinelibrary.com).
rheumatologic diseases [1]. Table II summarizes salient DOI: 10.1002/ajh.22023

V
C 2011 Wiley-Liss, Inc.

American Journal of Hematology 500 http://wileyonlinelibrary.com/cgi-bin/jhome/35105

 test of the month
TABLE I. Cryoglobulin Classification TABLE II. Clinical and Laboratory Features of Cryoglobulinemia

Cryoglobulin Clinical manifestations Type I Type II Type III

type Components Disease association
Arthralgia  arthritis 1 11 111
Type I Monoclonal immunoglobulins Waldenstrom’s Purpura 1, usually 111, 111,
(simple) (IgG, IgM, or IgA) or Bence macroglobulinemia nonpalpable palpable palpable
Jones protein/monoclonal Multiple myeloma Gangrene/acrocyanosis 111 1 ±
free light chains Monoclonal gammopathy Hyperviscosity 111 ± 2
associated with Hematologic 11 ± ±
lymphoproliferative disorder Renal 1 11 1
Light chain disease Neurologic 1 11 11
Type II Monoclonal immunoglobulins Hepatitis C Liver ± 11 111
(mixed) (IgM, IgG, or IgA) and Essential cryoglobulinemia Lung 2 1 1
polyclonal immunoglobulin Sjogren’s syndrome Cryocrit >5% <5% (1–2) <5% (1–2)
(mostly IgG) Rheumatoid arthritis C3 Normal Decreased Decreased
Chronic lymphocytic leukemia C4 Normal Decreased Decreased
Type III Polyclonal immunoglobulins Essential cryoglobulinemia CH50 Normal Decreased Decreased
(mixed) of all isotypes Sjogren’s syndrome RF 1 11 11
Systemic lupus erythematosus Autoantibodies (ANA, 2 11 11
Viral infections (Hepatitis B ENA, AMA, etc.)
and C, CMV, EBV, HIV) Hepatitis B 2 1 1
Endocarditis, other Hepatitis C 2 111 111
bacterial infections
Biliary cirrhosis RF, rheumatoid factor; ANA, antinuclear antibodies; ENA, antibodies to extracta-
ble nuclear antigens [such as Sm, RNP, Ro (SS-A), La (SS-B), Jo-1, Scl-70)];
CMV, cytomegalovirus; EBV, Epstein-Barr virus AMA, antimitochondrial antibodies. Hematologic features include thrombosis and
Adapted from Refs. 6 and 7. bleeding and spurious/artifact thrombocytosis. Adapted from Refs. 1,2,10–13.

increases the temperature at which the precipitate forms. amount of cryoglobulins may be pathogenic, therefore the
The three serum containing tubes are then kept at 48C and immunophenotype as well as the clinical context are important
analyzed at 72 hr. Type I cryoprecipitate may appear as in deciding the clinical relevance of cryoglobulin test results.
early as 24 hr, but mixed cryoglobulins may precipitate after Difficulties with the current testing technique for cryoglo-
several days; therefore, some laboratories wait until 7 days bulins include the requirement of large sample volume (10–
if there is no precipitate formed initially. 15 cm3) and the time to analysis of 3 or more days. More-
After visual observation, the precipitate formed at 48C over, there are no standards and controls available for the
can be reported in one of three ways: (1) cryocrit (percent cryoglobulin test, including no widely used reference ranges
of total volume), (2) protein content, or (3) immunoglobulin for cryocrit protein content or immunoglobulin content values
content [10]. There is no widely accepted reference range [7,10]. Visual inspection of cryoprecipitate is less accurate at
for cryoglobulins. Using protein content for reference values low concentrations of cryoglobulins, therefore improving sen-
is complicated by wide variation of total protein content in sitivity and specificity of cryoglobulin testing is an important
cryoprecipitate possibly due to coprecipitation of other pro- topic as in most cases the concentration of cryoglobulins do
teins. Reference values for individual immunoglobulins in not have to directly correlate with intensity of symptoms [10].
the cryoprecipitate have been described [11], but cryopreci- Standardized testing technique is important to isolate cryo-
pitate protein and immunoglobulin contents are rarely globulins which are present in low concentrations compared
reported by clinical laboratories. The most practical and to normal serum protein [7]. Interestingly, some healthy indi-
clinically useful are the cryocrit values in a longitudinal viduals can have detectable levels of cryoglobulins with an
analysis of patient’s clinical outcome. unknown significance. Given the variability of testing condi-
If there is a precipitate formed at 72 hr at 48C, the precipi- tions used in different laboratories and the lack of test stand-
tate is washed and recovered in cold phosphate-buffered sa- ards and reference values, further investigation into stand-
line. Washing of the sample at 48C facilitates removal of the ardization of the cryoglobulin testing should be performed in
contaminating normal serum proteins, thus allowing analysis the future. The biological importance and activity of cryoglo-
of the cryoglobulin exclusively. The cryoprecipitate is solubi- bulins such as its ability to activate proinflammatory comple-
lized at 378C and analyzed by immunodiffusion and immu- ment proteins needs to be defined as well.
nofixation to identify the cryoglobulin type. Many laborato- Treatment of Cryoglobulinemia
ries use antibodies to IgG, IgA, IgM, kappa or lambda as Treatment of Type I cryoglobulinemia focuses on
well as to C3 and C4 to identify the immunoreactants. The decreasing the formation of the cryoprecipitable monoclonal
clonality is determined by immunofixation electrophoresis. proteins. Prewarming blood transfusions, steroids, less
Incorporating anti-albumin antibodies in the analysis serves commonly splenectomy (for idiopathic cases) and treatment
to ensure quality of the washing of the cryoglobulins and of the underlying lymphoproliferative disorder are some of
confirmation of removal of contaminating serum proteins. the treatment options. In patients with hyperviscosity, plas-
False negative results can occur due to the following rea- mapheresis and cytotoxic agents are useful. Treatment of
sons. (1) Improper handling of the patient’s sample: If the Types II and III cryoglobulinemia includes treatment of
sample is not transported at 378C before processing, cryopre- infections such as HCV, steroids, other immunosuppressant
cipitate may form in transit during clotting of the sample, agents, plasmapheresis (especially for patients with hyper-
thereby decreasing the chance of cryoglobulin recovery. viscosity syndrome), cytotoxic chemotherapy, and rituximab
Thus, proper collection, optimum clotting, and centrifugation [12–17]. Plasmapheresis has a limited role but can be use-
are essential to avoid false negative results. (2) Storage of the ful in the acute phase. Low antigen diet (avoidance of mac-
sample at a temperature higher than 48C may impede or romolecular antigen containing foods such as diary prod-
abrogate cryoprecipitate formation and contribute to false ucts, meats and eggs, and gluten) has been used in some
negative results. (3) Lipemia in a patient’s sample causes se- patients, improving the function of the reticuloendothelial
rum turbidity due to lipids and may also interfere with correct system thereby possibly improving clearance of immune
cryoglobulin identification. Positive results of 1% or more of complexes. Symptoms of polyarthritis may respond to ste-
cryocrit are abnormal but in some cases even a minute roids, hydroxychloroquine, or cyclosporine for more severe

American Journal of Hematology 501

test of the month
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usually guided by improvement in symptoms, decrease in Arthritis Rheum 2004;33:355–374.
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rheumatoid factor activity. tice in the detection, analysis, and reporting of cryoglobulins. Clin Chem
2008;54:39–43.
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