Cryoproteins are proteins that, below body temperature, exhibit precipitation phenomena that often cause diseases. There is no automated analysis, at the click of a button on some routine devices, and there are no manufacturers of diagnostic tests that make money from or advertise cryoprotein diagnostics. In fact, the low reimbursement leads to a kind of avoidance strategy when it comes to medical examinations. Therefore, cryoproteins are often not detected, or are only detected much later, because the routine approach to taking blood samples and analyzing the serum parameters is not conducive to their discovery. An understanding of the medical and laboratory practice is important in order for those involved in taking blood samples and laboratory testing to cooperate with each other to prevent an early cryoprecipitation, render the antibodies visible and thus to draw the proper diagnostic conclusions. Otherwise, the process will result in frustration, mutual recrimination and incorrect analytical findings. The organizational effort is justified by the diagnostic value. The reward for such a process is a patient’s correct diagnosis, whose illness may have been misunderstood for far too long. Conversely, the positive detection of cryoproteins is a laboratory finding that must trigger further analysis of the conditions described below, if they are not already known. Our daily practice of dealing with laboratory staff and senders as well as, in our experience, the very rare request , led us to present and review the facts around cryoproteins, even though they have been dealt with in standard works [2–4]. Through improved technology and resolution in immunophenotyping, a relatively high association with non-Hodgkin’s lymphoma has been discovered particularly for cold antibodies and cryofibrinogen [5, 6].
The terms in this field are often used imprecisely and are misunderstood. One must distinguish the following terms:
Cryoglobulins are immunoglobulins (antibodies) in human serum, which at reduced temperature (<37 °C) precipitate spontaneously (Figure 1), and usually dissolve when heated again. These precipitates can occur in the patient (in vivo), but also after taking a blood sample (in vitro). The precipitation behavior of these proteins varies from patient to patient, and even over time in the same patient, depending, among other factors, on the concentration, immunoglobulin class, amino acid sequence, body temperature, outdoor temperature and pH value. In vivo, they naturally occur frequently in the colder months and may become noticeable on account of painful circulatory disorders of the skin. In the case of Waldenstrom’s disease (immunocytoma, lymphoma with secretion of IgM, rarely IgG), the IgM antibodies can cause a hyperviscosity syndrome. However, this can also occur with a multiple myeloma. In adults, cryoglobulins occur with high incidence in connection with hepatitis B or C, autoimmune diseases or lymphoma with immunoglobulin secretion, and in children rarely after a virus infection, with or without additional cold antibodies .
Cryofibrinogen is an insoluble (when it is cold) clot in human EDTA plasma made up of fibrinogen, fibrin, fibrin degradation products, thrombin, immunoglobulins, albumin and other proteins that forms at 4 °C and usually dissolves again at 37 °C. Cryofibrinogen can occur together with or independently of cryoglobulins. The distinction between cryofibrinogen and cryoglobulins is usually not clinically relevant. Cryofibrinogenemia is the underdiagnosed increase of a cryoprotein, which can be life threatening. Skin necrosis and thrombotic events are the main complications. Cryofibrinogenemia can be divided into two types: a primary (essential) and a secondary form associated with autoimmune diseases, malignancies and sepsis.
Cryoprecipitate formation in citrate-anticoagulated plasma is a physiological process used in the industrial fractionation of blood plasma proteins. After deep-freezing and slow thawing at 4 °C of citrate plasma, a reversible precipitate is formed that is rich in the proteins important for blood clotting: Factor VIII, von Willebrand factor and fibrinogen.
Cold (auto)antibodies are immunoglobulins directed against erythrocytes, almost exclusively of the IgM class (more rarely also against platelets or leukocytes), which cause the agglutination of the cells and which often, but not always, can be dissolved by heat. As these are specifically directed against red cell antigens and, as a rule, effectively bind and activate complement after the antigen-antibody reaction, the damage caused to the target cells by complements is not reversible when heated and leads in vivo and in vitro to cell destruction (hemolysis). They can occur together with or independently of cryoglobulins. The distinction between cryoglobulins and cold autoantibodies is of clinical relevance.
In the hematological laboratory, cryoglobulins can (!) irritatingly, appear as spherical precipitates, which are counted by the devices as platelets and, in aggregate form (Figure 2), as leukocytes, or the serum gels upon cooling. The precipitates are not necessarily spherical, but are characteristic if they are. This must be recognized as such and they are sometimes the first indication of the presence of cryoglobulinemia in patients.
A look at the blood smear is groundbreaking. Figures 3A–C show the cryoprecipitates from different patients in the smear. However, synthetic immersion oils can dissolve the precipitates again or cause them to become faint, so that they then escape the untrained observer, and finally become invisible. In the case of poor dyeing, the contrast has to be increased by closing the luminous-field diaphragm of the microscope and/or the gamma setting of the digital camera. The precipitates can significantly distort the platelet count as measured by impedance or optically. Alternative methods, such as a chamber count by an experienced lab worker using a microscope or the immunological platelet determination by means of a fluorescent monoclonal antibody against the GpIIb/IIIa receptor (CD61), allow for a correct analysis of the true concentration .
How does one recognize the presence of interference caused by cryoglobulinemia? It is important to observe the distribution curves (histograms) on the screen or on the printout from the hematology unit, to compare previous readings (delta check) and re-measure again after heating at 37 °C. Again, correct hematological values in these patients can be determined only if the sample was taken in a preheated tube (!) and kept warm during transport.
In serum analysis, the precipitates are usually overlooked because the blood is centrifuged and the precipitates remain in the clotted blood. As a result, they cannot be measured (total protein, immunoglobulins, serum electrophoresis, immunofixation electrophoresis). Cryoglobulins are not just an interfering artifact in the laboratory, but also point the way in diagnostic findings. When blood is sampled, transported and processed properly at the laboratory (heat chain at 37 °C, serum formation in the incubator >60 min, or ideally 2 h), the cryocrit can be measured and the precipitate analyzed for monoclonality of immunoglobulins. In some cases, it may also be cryofibrinogen (specific detection in EDTA blood).
Blood collection and pre-analysis
The correct blood collection in connection with a suspected cryoproteinemia is done via blood collection systems pre-heated to at least 37 °C (serum and EDTA tubes and needle systems stored in the practice or laboratory overnight in the incubator at 37–39 °C), with the blood sample being rushed to the laboratory in pre-heated containers (collection system and insulated containers pre-heated in the incubator). Undefined hot water cups, open heating pads, etc. are not helpful. Centrifugation must be carried out in a preheated centrifuge, followed by a direct measurement in the device (STAT-position without any wait time in the unit). Only then, is a correct analysis possible. Alternatively, the warm centrifugation can be done at the practice prior to sending the sample to the laboratory, provided the conditions can be maintained.
However, this means that the patient needs to be instructed and be called in separately. The blood collection must also be prepared, and this process takes up more time. The laboratory must be informed in advance. A deviation from the described ideal conditions leads to the risk of false negative findings. When taking a regular blood sample, a remote laboratory is hardly in the position to meet these requirements, so that the correct findings can be guaranteed. Therefore, the best solution will often be to send the patient to the laboratory for the blood collection.
The analytical investigation is well-described elsewhere [9–11]. The serum, consistently at 37 °C, is transferred to a cryocrit tube at the laboratory (a Wintrobe tube, see Figure 4, according to the American hematologist Maxwell Wintrobe VWR, Darmstadt, FRG). The scale on the tube is first read after 24 h and then again, after having been stored in the refrigerator for a week, at a temperature of 4 °C. The precipitation can sometimes take a long time (see Table 1). The glass tube must be rinsed without detergents, as detergents prevent precipitation.
At the same time, the same procedure is carried out for the detection/exclusion of cryofibrinogen on EDTA plasma.
With a positive finding and after the reading, it is checked whether the existing precipitate dissolves again when heat is applied (usually not the case with cryoglobulins). A control serum, always present, of a healthy donor does not show any precipitate (the serum must not freeze in the refrigerator!).
For this purpose, the precipitate is washed three times in ice-cold saline solution and examined in the immunofixation electrophoresis with the addition of solvents (e.g. 100 μL Fluidil (Sebia, Lisses, France)+1 μL ß-mercaptoethanol (Sigma-Aldrich, Munich, FRG) in deviation from the normal procedure of immunofixation). In this process, the immunoglobulin class and any existing monoclonality are determined. This allows for the classification of cryoglobulins, which may provide clues about the cause (see Table 1).
A medical practice can easily detect the cryoproteins if a suitable centrifuge with temperature control is available. The further necessary diagnostics must then be done at a specialist laboratory.
The clinical picture
Cryoglobulins are immunoglobulins (antibodies) in human serum. They represent not only an interfering laboratory artifact, but may also be active in vivo and cause multi-organ damage. In addition to the triad described by Meltzer of purpura, weakness and arthralgias, there are varying manifestations on the skin, in the joints, kidneys, nervous system and in hematopoiesis. The clinical treatment of these patients remains difficult. It focuses on the underlying lymphoproliferative, infectious disease or systemic cause. It comprises corticosteroids, other immunosuppressive agents and plasmapheresis. More recently, mostly rituximab, a monoclonal antibody to B-cells, has been used in order to interrupt the B-cell response.
Most frequently, cryoglobulins are encountered with monoclonal gammopathies, occasionally with chronic polyarthritis, glomerulonephritis, endocarditis lenta, syphilis, malaria, HCV infection, non-Hodgkin’s lymphomas (see Table 2). The cryoproteins can precede the overt disease by years.
Cryoglobulinemias are divided into i) monoclonal cryoglobulinemias (= type I), where a single type of monoclonal immunoglobulin is present, in which an autonomous juxtaposition occurs, and mixed cryoglobulinemias, which are characterized by ii) a mixture of either polyclonal immunoglobulin (Ig) G and monoclonal IgM (type II), or iii) polyclonal IgG and polyclonal IgM (= type III); both – monoclonal and polyclonal IgM – exhibit rheumatoid factor activity, i.e. precipitation is triggered by binding of the antibody to the Fc region of another antibody.
Cryoglobulinemia is a model disease for several reasons:
Cryoglobulins can be diagnosed by a technically simple test based on laboratory observation of precipitation in cold serum.
Organ damage resulting from cryoglobulinemia can be caused in two ways: accumulation of cryoglobulins and autoimmune-mediated vasculitic damage.
Cryoglobulinemia is associated with a wide spectrum of etiologies, symptoms and final stages and is considered a disease that combines the elements of autoimmune and lymphoproliferative diseases.
The clinical manifestations result from either the hyperviscosity syndrome or the vasculitis of small vessels. Although the hepatitis C virus (HCV) is a well-known factor in the etiology of cryoglobulinemia, there are substantial geographic differences in the association between cryoglobulins and HCV.
Cryofibrinogen was first described as a cryoprotein in 1955. Its prevalence is estimated at 0–7%; in hospitalized patients, the levels are said to be higher but very few data exist. Systemic manifestations are common and in addition to symptoms similar to cryoglobulinemia, arterial and venous thromboembolism may also occur. Mild forms are reversible, which are treated with corticosteroids and aspirin; in the case of thrombosis, with anticoagulants . Cryofibrinogenemia may occur as essential or secondary in the context of neoplasia or infections, thrombosis or tissue diseases with vasculitis. In approx. 50% of cases where cryofibrinogenemia is considered essential at first, underlying lymphomas (6 T- and 5 B-cell lymphomas) were found over time, as a study has shown , whether as a cause or effect, which is why patients need to be monitored after diagnosis.
Essential cryofibrinogenemia represents approximately 12% of all cryoproteins. Thrombotic events are frequent and correlated with the amount of plasma-cryofibrinogen. A defect in fibrinolysis may lead to an accumulation of cryofibrinogen and clotting in small and medium-sized arteries. In some cases initially classified as essential, lymphomas were the cause and should therefore be specifically looked for.
Diagnostic criteria for essential cryofibrinogenemia (from: Amdo and Welker )
Typical clinical presentation: Sudden onset with skin lesions and constitutional symptoms with or without thrombosis, hemorrhage or exposure to cold
Presence of cryofibrinogen in plasma
Absence of cryoglobulins
No secondary causes for cryofibrinogens and no evidence of other vascular occlusive diseases
Angiogram with abrupt occlusion of small to medium-sized arteries
Typical findings in skin biopsy: vessels clogged by cryofibrinogen, leukocytoclastic vasculitis or skin necrosis
Increased serum levels of alpha1-antitrypsin and alpha2-macroglobulin
Differential diagnoses for essential cryofibrinogenemia (from: Amdo and Walker )
Peripheral vascular disease
Thrombotic thrombocytopenic purpura (TTP, Moschcowitz syndrome)
Disseminated intravascular coagulation (DIC)
Hereditary states of hypercoagulability
Embolic diseases such as endocarditis or cholesterol emboli
Calciphylaxis in end-stage renal disease
Antiphospholipid antibody syndrome
Example for the frequency of cryofibrinogenemia from a study by Saadoun et al. : In 10 years, 2312 tests were requested at a university clinic, of which 515 were positive (22%), using a cutoff of 50 mg/L, at two different points in time; 455 were associated with cryoglobulinemia (88%). Of the remaining 60 cases of an isolated cryofibrinogenemia, 36 (60%) were essential and 24 (40%) secondary.
Cold antibodies against erythrocytes are a differential diagnosis of hemolytic anemias and are detected by means of the direct Coombs test (direct antiglobulin test, DAT) in acute hemolysis with good sensitivity. In childhood, they are mainly infection-associated, but adults with mycoplasma infections were also DAT-positive in 57% of the cases . In addition, cold autoantibodies represent the central pathophysiologic principle in all forms of cold autoimmune hemolysis and cold agglutinin disease; accompanying cold autoantibodies in B-cell lymphomas are not rare, and the screening via the direct Coombs test, usually recognizable by C3d-loading, makes sense. This association with B-cell lymphoma, which is ascribed a separate entity apart from the immunocytoma , leads to new therapeutic options, the use of CD20 B-cell antibody rituximab with additional administration of fludarabine [15–18]. This is how cold antibodies became tumor markers.
Cryoproteins, in contrast to hemolysis, icterus and lipemia, are not really analyzed systematically in everyday laboratory practice and are often only tested for by blood banks. There is a discrepancy between the frequency of systematic search in the context of research studies and daily practice. Each laboratory should therefore check if it wants to include these investigations in its program of available tests, because of the necessity of this test, for example, in connection with clarifying a thrombosis diagnosis at an coagulation clinic, a skin clinic or rheumatism clinic. If a laboratory arrives at an incidental finding of cryoproteins or cold agglutinins, the sender should definitely be alerted in a clear manner. Despite centrifugation, cryoglobulins may become visible with a delay (storage of samples in the cooler for re-analyses). Tubes stored for over 1 to 2 weeks, a standard in most labs, should be inspected before discarding them. When doing a blood count, and especially in smears, attention should be paid to cryoproteins, which should be noted in the findings. Table 3 summarizes what has been described.
Shihabi ZK. Cryoglobulins: an important but neglected clinical test. Ann Clin Lab Sci 2006;36:395–408.Google Scholar
Thomas L. Labor und Diagnose: Indikation und Bewertung von Laborbefunden für die medizinische Diagnostik, 6th ed. Frankfurt/Main: TH-Books Verlagsgesellschaft mbH, 2005:978–81.Google Scholar
Deutsch E, Geyer G, Wenger R. Laboratoriumsdiagnostik. 3rd ed. Basel: S. Karger Verlag, 1992:840–1.Google Scholar
Keil E, Fiedler H. Klinische Chemie systematisch. 1st ed. Bremen: UNI-MED Verlag AG, 2000:265.Google Scholar
Randen U, Troen G, Tierens A, Steen C, Warsame A, Beiske K, et al. Primary cold agglutinin-associated lymphoproliferative disease: a B-cell lymphoma of the bone marrow distinct from lymphoplasmacytic lymphoma. Haematologica 2014;99: 497–504.Web of ScienceGoogle Scholar
von Ahsen N, Ehrlich B, Scott CS, Riggert J, Oellerich M. Cryoglobulins interfere with platelet counts by optical and impedance methods but not with the CD61 immunoplatelet count. Clin Chem 2001;47:1858–60.Google Scholar
Kallemuchikkal U, Gorevic PD. Evaluation of cryoglobulins. Arch Pathol Lab Med 1999;123:119–25.Google Scholar
Thomas L. Labor und Diagnose: Indikation und Bewertung von Laborbefunden für die medizinische Diagnostik, 5th ed. Frankfurt/Main: TH-Books-Verlagsgesellschaft mbH, 1998:689–99.Google Scholar
Belizna C, Loufrani L, Subra JF, Godin M, Jolly P, Vitecocq O, et al. A 5-year prospective follow-up study in essential cryofibrinogenemia patients. Autoimmun Rev 2011;10:559–62.Web of ScienceCrossrefGoogle Scholar
Amdo TD, Welker JA. An approach to the diagnosis and treatment of cryofibrinogenemia. Am J Med 2004;116:332–7.Google Scholar
Ali NJ, Sillis M, Andrews BE, Jenkins PF, Harrison BD. The clinical spectrum and diagnosis of Mycoplasma pneumoniae infection. Q J Med 1986;58:241–51.Google Scholar
Original German online version at: http://www.degruyter.com/view/j/labm.2014.38.issue-5/labmed-2014-0033/labmed-2014-0033.xml?format=INT. The German article was translated by Compuscript Ltd. and authorized by the authors.