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
below 378C from patient’s serum and redissolve when
rewarmed in vitro [1,2]. They were first observed in 1933
by Wintrobe and Buell [3] in a patient with Raynaud’s phe-
nomenon. They were called cryoglobulins (cold precipitable
serum globulin) in 1947 [4]. In the 1960s, a clinical profile
of palpable purpura, asthenia, arthralgia, and renal disease
was recognized, caused by immune complex deposition
and cryoglobulinemia [5]. There are three types of cryoglo-
bulins depending on their characteristics and interactions
with other proteins (Table I) [6].
The laboratory workup of suspected cryoglobulin should
be performed in patients with unexplained multiorgan dis-
ease involvement such as skin, liver, renal, peripheral nerve
manifestations, Raynaud’s syndrome, and in patients with
the typical triad (first described by Meltzer and hence
known as Meltzer’s triad) of purpura, weakness, and
arthralgias [5].
Monoclonal cryoglobulinemia (Type I) is often seen in
patients with hematologic malignancies and can cause
hyperviscosity syndrome. Mixed cryoglobulinemia (Types II
and III) often presents as a systemic vasculitis due to
immune complex deposition in the small vessels and may
be the presenting feature in patients with infectious and
systemic disorders [7]. Patients may present with palpable
purpura, weakness, arthralgia as well as liver and renal
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
patient population) with mixed cryoglobulinemia have Hepa-
titis C infection (HCV) versus 1.8% having Hepatitis B [8,9].
Conversely, about 40–66% of patients with HCV have ele-
vated circulating monoclonal and/or polyclonal immunoglo-
bulins. The clinical syndrome of cryoglobulinemia is mani-
fested in only about 5% of HCV patients [7]. Among the
Type III cryoglobulins, the mixed cryoglobulinemia can be
associated with organ involvement and small and medium
size vessel vasculitis and is again seen in infectious and
rheumatologic diseases [1]. Table II summarizes salient
syndromes.
Laboratory Testing for Cryoglobulins
For cryoglobulin identification, two 10 mL red top tubes
of sample (without an anticoagulant) are drawn from a
patient and transported to the laboratory at 378C (com-
monly, this is achieved by submerging the tubes in warm
water during transportation to the laboratory). The sample
is allowed to clot at 378C for 1 hr. The clot is separated
from the sides of the tube with a Pasteur pipette, and the
serum is separated by warm (378C) centrifugation and ali-
quoted into three tubes at 378C. One tube is the Wintrobe
tube for quantification of the cryoprecipitate (a Wintrobe
tube has markings that are used to quantify). The second
tube contains a larger quantity of serum, which is used for
cryoprecipitate observation and subsequent analysis. The
third tube is used to determine the solubility of the cryopre-
cipitate on rewarming to 378C, a feature of cryoglobulin. If
a warm centrifuge is not available, serum should be sepa-
rated in a 378C water bath after allowing cells to clot and
settle at 378C for an hour without centrifugation, being
careful to ensure that the red cells and fibrin clot are not
aspirated with the serum. It is important for the sample
temperature not to drop below 378C until serum separation
is complete to avoid premature precipitation. Improper sam-
ple handling may result in a missed diagnosis of cryoglobu-
linemia [10]. Increasing concentration of cryoglobulins
Conflict of interest: Nothing to report.
1
Massachusetts General Hospital Cancer Center, Boston, Massachusetts;
2
Dana-Farber Cancer Institute, Boston, Massachusetts; 3Department of
Medicine, Massachusetts General Hospital, Boston, Massachusetts
*Correspondence to: Mandakolathur Murali, Clinical Immunology Laboratory,
Massachusetts General Hospital, 55 Fruit Street, Boston, Massachusetts
02114. E-mail: mmurali@partners.org
Received for publication 11 February 2011; Revised 23 February 2011;
Accepted 26 February 2011
Am. J. Hematol. 86:500–502, 2011.
Published online 14 March 2011 in Wiley Online Library (wileyonlinelibrary.com).
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.
The three serum containing tubes are then kept at 48C and
analyzed at 72 hr. Type I cryoprecipitate may appear as
early as 24 hr, but mixed cryoglobulins may precipitate after
several days; therefore, some laboratories wait until 7 days
if there is no precipitate formed initially.
After visual observation, the precipitate formed at 48C
can be reported in one of three ways: (1) cryocrit (percent
of total volume), (2) protein content, or (3) immunoglobulin
content [10]. There is no widely accepted reference range
for cryoglobulins. Using protein content for reference values
is complicated by wide variation of total protein content in
cryoprecipitate possibly due to coprecipitation of other pro-
teins. Reference values for individual immunoglobulins in
the cryoprecipitate have been described [11], but cryopreci-
pitate protein and immunoglobulin contents are rarely
reported by clinical laboratories. The most practical and
clinically useful are the cryocrit values in a longitudinal
analysis of patient’s clinical outcome.
If there is a precipitate formed at 72 hr at 48C, the precipi-
tate is washed and recovered in cold phosphate-buffered sa-
line. Washing of the sample at 48C facilitates removal of the
contaminating normal serum proteins, thus allowing analysis
of the cryoglobulin exclusively. The cryoprecipitate is solubi-
lized at 378C and analyzed by immunodiffusion and immu-
nofixation to identify the cryoglobulin type. Many laborato-
ries use antibodies to IgG, IgA, IgM, kappa or lambda as
well as to C3 and C4 to identify the immunoreactants. The
clonality is determined by immunofixation electrophoresis.
Incorporating anti-albumin antibodies in the analysis serves
to ensure quality of the washing of the cryoglobulins and
confirmation of removal of contaminating serum proteins.
False negative results can occur due to the following rea-
sons. (1) Improper handling of the patient’s sample: If the
sample is not transported at 378C before processing, cryopre-
cipitate may form in transit during clotting of the sample,
thereby decreasing the chance of cryoglobulin recovery.
Thus, proper collection, optimum clotting, and centrifugation
are essential to avoid false negative results. (2) Storage of the
sample at a temperature higher than 48C may impede or
abrogate cryoprecipitate formation and contribute to false
negative results. (3) Lipemia in a patient’s sample causes se-
rum turbidity due to lipids and may also interfere with correct
cryoglobulin identification. Positive results of 1% or more of
cryocrit are abnormal but in some cases even a minute
amount of cryoglobulins may be pathogenic, therefore the
immunophenotype as well as the clinical context are important
in deciding the clinical relevance of cryoglobulin test results.
Difficulties with the current testing technique for cryoglo-
bulins include the requirement of large sample volume (10–
15 cm3) and the time to analysis of 3 or more days. More-
over, there are no standards and controls available for the
cryoglobulin test, including no widely used reference ranges
for cryocrit protein content or immunoglobulin content values
[7,10]. Visual inspection of cryoprecipitate is less accurate at
low concentrations of cryoglobulins, therefore improving sen-
sitivity and specificity of cryoglobulin testing is an important
topic as in most cases the concentration of cryoglobulins do
not have to directly correlate with intensity of symptoms [10].
Standardized testing technique is important to isolate cryo-
globulins which are present in low concentrations compared
to normal serum protein [7]. Interestingly, some healthy indi-
viduals can have detectable levels of cryoglobulins with an
unknown significance. Given the variability of testing condi-
tions used in different laboratories and the lack of test stand-
ards and reference values, further investigation into stand-
ardization of the cryoglobulin testing should be performed in
the future. The biological importance and activity of cryoglo-
bulins such as its ability to activate proinflammatory comple-
ment proteins needs to be defined as well.
Treatment of Cryoglobulinemia
Treatment of Type I cryoglobulinemia focuses on
decreasing the formation of the cryoprecipitable monoclonal
proteins. Prewarming blood transfusions, steroids, less
commonly splenectomy (for idiopathic cases) and treatment
of the underlying lymphoproliferative disorder are some of
the treatment options. In patients with hyperviscosity, plas-
mapheresis and cytotoxic agents are useful. Treatment of
Types II and III cryoglobulinemia includes treatment of
infections such as HCV, steroids, other immunosuppressant
agents, plasmapheresis (especially for patients with hyper-
viscosity syndrome), cytotoxic chemotherapy, and rituximab
[12–17]. Plasmapheresis has a limited role but can be use-
ful in the acute phase. Low antigen diet (avoidance of mac-
romolecular antigen containing foods such as diary prod-
ucts, meats and eggs, and gluten) has been used in some
patients, improving the function of the reticuloendothelial
system thereby possibly improving clearance of immune
complexes. Symptoms of polyarthritis may respond to ste-
roids, hydroxychloroquine, or cyclosporine for more severe

American Journal of Hematology 501

test of the month
conditions [2]. The treatment course for cryoglobulinemia is
usually guided by improvement in symptoms, decrease in
cryocrit, restoration of complement activity, and decrease in
rheumatoid factor activity.
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