Cryoglobulin pre-analysis over the weekend

To the Editor,

Maintenance of blood samples at 37 °C during all pre-analytical phases is crucial for optimal cryoglobulin detection. Because the pre-analytical cryoglobulin workflow is laborious, time-consuming and requires specialized equipment, most hospitals do not accept blood samples for cryoglobulin analysis during weekends or bank holidays. In this validation study we demonstrated that during pre-analytical cryoglobulin workup the clotting time can be extended up to three days when blood samples are stored at 37 °C, allowing further cryoglobulin analysis during office hours.

Cryoglobulins consist of serum immunoglobulins that precipitate below 37 °C and resolubilize upon warming. Cryoglobulinemia generally involves small-to-medium vessel vasculitis caused by cryoglobulin-containing immune complexes. Cryoglobulinemia can cause a broad variety of symptoms including purpura, livedo reticularis, ischemia of extremities, arthralgia, fatigue, peripheral neuropathy, abdominal pain and glomerulonephritis. Treatment options are primarily directed towards identification and management of the underlying disease [1]. Fifty years ago Brouet et al. proposed a classification of cryoglobulins [2], which is still widely used because it is indicative for the identification of the disorder that underlies the cryoglobulinemia. Type I cryoglobulins are monoclonal immunoglobulins. Type II cryoglobulins are composed of both a monoclonal and polyclonal immunoglobulins. Type III cryoglobulins are composed of exclusively polyclonal immunoglobulins [3].

The laboratory work-up of cryoglobulin diagnostics involves three phases [4]. In the pre-analytical phase it is crucial that the blood remains at 37 °C throughout all steps from drawing blood, transport, coagulation and the centrifuge step to separate serum. In the second phase the serum is incubated for at least three days (preferably seven) at 4 °C to allow cryoglobulins to precipitate. The formed cryoglobulin precipitate is washed in saline (0.9 % NaCl), resolubilized at 37 °C and quantified. Finally, the washed cryoprecipitate is typed using immunofixation electrophoresis. The most critical factor affecting cryoglobulin testing is that all pre-analytical steps are completed at 37 °C [5], [6]. This unique pre-analytical workflow requires amongst others specialized equipment such as a pre-heated centrifuge which is not easily implemented in a 24/7 diagnostic service. This is one of the reasons why most hospitals can’t accept blood samples for cryoglobulin analysis during weekends or bank holidays.

It is well established in literature that collected blood for cryoglobulin analysis should coagulate at 37 °C for at least 1 h prior to separation of the serum-fraction by centrifugation [3], [5], [7]. It has never been studied whether the coagulation time at 37 °C can be extended with a couple of days. This would be valuable information in case a medical center is willing to accept cryoglobulin blood samples any day and hour of the week and postpone further pre-analytical workup to regular office hours. In this study we therefore validated whether blood samples for routine cryoglobulin testing could be stored and clotted at 37 °C for 72 h prior to further serum processing.

For this method comparison two different pre-analytical workflows were compared: ‘routine’ cryoglobulin analysis vs. our adapted ‘3-day-protocol’. For routine cryoglobulin analysis we have adopted the protocol previously described by Sargur et al. [7]. Briefly, after collection of serum blood-tube without separating gel it is directly transported in a preheated container to the laboratory at 37 °C. The sample is allowed to clot for 1–2 h at 37 °C and centrifuged 1,500 g for 15 min at 37 °C. At least 4 mL serum is separated and stored for at least three days at 4 °C. After centrifugation at 1,500 g for 15 min at 4 °C, visual inspection is performed to identify cryoprecipitate formation. Positive samples are washed three times at 1,500 g for 15 min with saline (0.9 % NaCl) at 4 °C. The washed cryoprecipitate is dissolved in 1 mL saline at 37 °C. Quantification of the cryoprecipitate is performed measuring the total protein concentration using an in-house Lowry protein assay. Cryoprecipitate typing is performed with immunofixation electrophoresis on a hydrasys-2 device (Sebia, Evry, France). For evaluation of our adapted ‘3-day-protocol’ we performed a second cryoglobulin analysis next to our routine cryoglobulin analysis in a total of 14 different patients. For the ‘3-day-protocol’ the exact same procedure was performed as described above with the only exception that the blood was allowed to clot at 37 °C for 72 h, instead of 1–2 h.

Detection of cryoprecipitation: When the blood samples were stored at 37 °C for 72 h prior to serum collection, referred to as the ‘3-day-protocol’, all sera became hemolytic which was not observed when the routine protocol was applied (Figure 1A). When the sera were stored for at least three days at 4 °C, the hemolysis did not affect visual interpretation of the cryoprecipitate formation. Visual inspection of both results of all 14 patients performed by two independent technicians did not show discrepancies between both pre-analytical protocols in any of the samples. Seven samples were classified as negative by both methods and seven samples were classified as positive by both methods. The pre-analytical protocol also did not affect the cryoprecipitate washing procedure nor its capacity to dissolve again at 37 °C (data not shown).

Figure 1:

Cryoglobulin analysis. (A) Five representative pictures taken of serum with a positive cryoglobulin obtained using our routine protocol (always left on picture) and using our ‘3-day-protocol’ (always right on picture). Hemolysis was observed in all sera obtained with the ‘3-day-protocol’ (average H-index 164). Approximately the same amount of cryoprecipitate was detected in both pre-analytical methods. (B) Immunofixation electrophoresis scans of one representative sample for each Brouet type is shown. The left scan shows the result when the routine pre-analytical protocol was used and the right scan shows the result when the ‘3-day-protocol’ was applied.

Cryo quantification: The washed crypreciptates have a whitish appearance in both pre-analytical protocols and no significant quantitative differences were observed (Table 1), indicating that the hemoglobulin and lysed erythrocytes do not end up in the washed cryoprecipitate. It must be noted however that quantification differences between both protocols is based on only seven positive samples. The small differences in cryoprecipitate concentration within individual patients are most likely caused by intra-assay variation of cryoprecipitate quantification since poor reproducibility of cryoglobulin quantification is a well described phenomenon [7].

Cryo typing: From the seven patients with a positive cryoglobulin result, the cryoprecipitates obtained with both the routine pre-analysis and the ‘3-day-protocol’ were all further processed for cryo typing using immunofixation electrophoresis. Independent interpretation of the results by two technicians resulted in 100 % identical Brouet classifications in all seven patients (Figure 1B). The quality of the cryoprecipitate washing step is amongst others assessed by looking at the background albumin band in the fixation-lane. Also in that aspect, no differences were observed between the two pre-analytical methods.

Drawing blood for cryoglobulin analysis is in most hospitals not feasible during weekends or bank holidays since pre-analysis requires specialized procedures that do not operate 24/7. A patient may therefore be asked to revisit the hospital for the sole purpose of undergoing a blood collection procedure for cryoglobulin analysis. In patients presenting to the emergency department during weekends with severe symptoms requiring immediate therapeutic intervention, obtaining a blood sample prior to the initiation of therapy may be hindered.

Here we showed that blood tubes drawn for cryoglobulin analysis could be stored and clotted at 37 °C for 72 h prior to serum separation in a pre-heated centrifuge. Although we observed signs of hemolysis in all samples that were measured with this ‘3-day protocol’, this did not affect the detection, immunotyping and quantification of cryoglobulins. The outcome of the data presented here, may facilitate simple and cost-effective solutions that allow drawing blood for cryoglobulin analysis during the weekend. For example, a plugged-in incubator [8] in the outpatient or emergency department allows stringent maintenance of pre-clotting temperature at 37 °C of blood samples obtained during the weekend. Directly after the weekend these blood samples can be collected and transported to the laboratory for further cryoglobulin analysis during office hours.