Abstract
Abstract
A 35-year-old man was found to have a negative LDL-cholesterol concentration (−0.05 mmol/L) when estimated on a fasting plasma sample using the Friedewald equation. Plasma urea, electrolytes and liver function tests (LFTs) were normal except for a raised total bilirubin of 74 μmol/L. Haematological results showed both a low haemoglobin and fibrinogen concentration. It transpired that the patient had undergone daily plasmapheresis treatments on the previous four days; plasma had been exchanged with a 5% albumin solution. He had been diagnosed with Evan's syndrome previously (characterized by autoimmune haemolytic anaemia) and had been admitted with severe anaemia, which had proved unresponsive to conventional treatments. The concentration of most plasma substances is reduced by 50–60% after one standard plasmapheresis treatment, with the rate of return to steady state concentrations varying among analytes. The finding of a negative LDL-cholesterol concentration (arising primarily as a result of normal triglyceride concentrations) may reflect the more efficient removal of LDL and HDL lipoproteins during the plasmapheresis procedure (PP) than lipoproteins containing proportionally more triglycerides. Plasma lipids, total protein, immunoglobulins and transferrin had recovered to steady state concentrations by eight days post-plasmapheresis, whereas caeruloplasmin concentrations had not. This case report illustrates the difficulties of obtaining accurate information on the steady state concentrations of plasma analytes, in particular protein bound substances, when analysis is carried out on a sample from a patient that has recently undergone plasmapheresis. The normal plasma albumin in this situation did not flag the possibility of the sample being artefactually diluted.
Case report
A 35-year-old man was found to have a negative LDL-cholesterol concentration when estimated on a fasting plasma sample using the Friedewald equation (LDL-cholesterol mmol/L = total cholesterol mmol/L – HDL-cholesterol mmol/L – triglyceride mmol/L/2.2). The plasma lipid results were as follows: total cholesterol = 1.0 mmol/L, triglycerides = 1.60 mmol/L, HDL-cholesterol = 0.32 mmol/L and LDL-cholesterol = −0.05 mmol/L. Plasma lipid analysis had been requested as this patient was being investigated for haemophagocytic syndrome, which is characterized by, among other findings, a normal plasma total cholesterol and raised triglyceride and ferritin concentrations. LDL-cholesterol had been estimated due to the fact that a full cholesterol profile is automatically requested on a fasting sample received for lipid analysis in our laboratory. Plasma urea and electrolytes were normal with creatinine slightly low at 47 μmol/L (62–106). Albumin, γ-glutamyl transferase (GGT) and alanine aminotransferase (ALT) concentrations were all within reference limits with total bilirubin being raised at 74 μmol/L (1–21) and alkaline phosphatase (ALP) low at 15 U/L (35–129). Abnormal haematological results included a low haemoglobin, 7.3 g/dL (13–18), low red cell count, 2.43 × 1012/L (4.5–6.5 × 1012), low platelets, 81 × 109/L (150–400 × 109) and low fibrinogen 0.58 g/L (1.50–4.00). The patient was being treated with steroids and antibiotics, but was not on any lipid-lowering medication or receiving parenteral nutrition. Biochemical and haematological test results from four days previously were as follows; plasma electrolytes and creatinine normal, urea 10.3 mmol/L (2.1–7.1), albumin 35 g/L (35–50), total bilirubin 417 μmol/L, ALP 60 U/L, GGT 144 U/L (8–61), ALT 327 U/L (4–50), haemoglobin 3.7 g/dL, red cell count, 0.99 × 1012/L and fibrinogen 3.5 g/L.
On further inquiry, it transpired that the patient had undergone four treatments of plasmapheresis (plasma exchange) daily for the four previous days. On each day, 4.5 L of plasma was exchanged with a 5% albumin solution in saline. Total protein measured on the sample above with the negative LDL-cholesterol concentration was low at 49 g/L (60–85) with individual plasma proteins (apart from albumin) being also low (Table 1). Two days later (three days post-plasmapheresis treatment), plasma electrolytes, urea, creatinine, albumin, GGT, ALP, total protein, haemoglobin and red cell count remained unchanged, with bilirubin decreasing to 37 μmol/L and ALT and transferrin increasing to 71 U/L and 1.59 g/L, respectively. Plasma concentrations of lipids had also increased (Table 1). As this was a non-fasting blood sample, the value for plasma triglycerides may be overestimated and hence the value for LDL-cholesterol may be underestimated as a result. Five days later (eight days post-plasmapheresis treatment) the patient's biochemical results had remained unchanged except for the ALT concentration, which had increased to 102 U/L, and total bilirubin and ALP concentrations that were now within reference limits. Haemoglobin had increased to 10 g/dL with the red cell count at 3.13 × 1012/L. Plasma protein and lipid concentrations had further increased in concentration, analysis again carried out on a non-fasting sample (Table 1). Subsequently, ALT concentrations declined and reached concentrations within reference limits after a further 10 days. The patient's anaemia gradually resolved, with the full blood count being normal one month after plasmapheresis. Results of plasma protein and lipid analysis performed on a fasting sample four months after plasmapheresis are illustrated in Table 1.
Plasma lipid and protein concentrations
*Reference range applies to fasting samples
PP, plasmapheresis procedure
The patient had been diagnosed with Evan's syndrome 1 at the age of 15 years. This syndrome is characterized by immune thrombocytopenia and autoimmune haemolytic anaemia caused by the production of autoantibodies against red blood cells and platelets. The patient had undergone a splenectomy at the age of 28 years and had been intermittently treated with steroids and intravenous immunoglobulin for his condition. On this occasion, he had been admitted to hospital the previous week with a haemoglobin of 7.0 g/dL, which had decreased to 4.0 g/dL after two days. He was treated with steroids, intravenous immunoglobulin and Rituximab (anti-B cell antibody) in an effort to reduce autoantibody production, but his haemoglobin remained at 4.0 g/dL even with multiple blood transfusions. Although the autoantibody in this condition is normally attached to red blood cells, because of the aggressive disease activity in this patient, overflow of the autoantibody had occured into the plasma. It was decided to treat this patient with plasmapheresis due to the critical nature of his condition. As stated above, he underwent plasmapheresis daily for four days and was also treated with steroids and received intravenous fibrinogen to replace that lost during the procedure. Within two weeks he had responded to red cell infusions and by seven weeks he was off all treatment (apart from prophylactic antibiotics due to his splenectomy). He has remained in remission for over four months.
Discussion
Plasmapheresis (also known as therapeutic plasma exchange) is a process by which blood is removed from the body and centrifuged to separate the blood cells from plasma.
2
It is usually performed by a continuous centrifugation technique whereby only 150 mL of blood is outside the body at any one time. It is a blood purification technique designed for the removal of large molecular weight substances (>15,000 Daltons) that cannot be efficiently removed by other techniques such as haemodialysis. After the separation of plasma, the blood cells are then returned to the patient together with either the plasma treated to remove the desired components, fresh frozen plasma or with a solution of saline with added proteins. The most common replacement fluid used is 5% albumin. The standard plasma volume exchanged during a single plasmapheresis treatment is a volume corresponding to the total plasma volume of the patient which is calculated from the patient's weight and haematocrit and varies between 3 and 5 L for the majority of patients. In general, large molecular weight substances (e.g. immunoglobulins, lipoproteins) are only slowly equilibrated between their extravascular and intravascular distribution (1–3% per hour) so that the expected plasma concentration (C1) after plasmapheresis can be determined by the application of first-order kinetics using the formula
2
;
The rate of return of plasma constituents to their steady state concentrations post-plasmapheresis is dependent on their rate of de novo synthesis (and/or ingestion), their rate of extravascular to intravascular redistribution and both the number and frequency of plasmapheresis treatments. 2 When albumin is used as the sole replacement fluid, the main clinical concern is a depletion of coagulation factors leading to an increase in clotting time and the removal of immunoglobulins and complement that may predispose the patient to a risk of infection. After multiple treatments over a relatively short period of time, it has been shown that the depletion in complement concentrations and clotting factors may require several days for spontaneous recovery 14,15 and that the decrease in immunoglobulin concentration (especially when combined with concomitant immunosuppressive therapy) may persist for several weeks. 15 However, replacement of coagulation factors and immunoglobulins can be instigated if considered necessary. Apart from complement, immunoglobulins, lipoproteins and coagulation factors, there is little information in the literature on the rate of the subsequent recovery of plasma concentrations of plasma analytes post-plasmapheresis. With regard to the biochemical data presented in this report, the finding of a negative LDL-cholesterol concentration (which can effectively be interpreted as a very low concentration), as a result of the application of the Friedewald formula, may reflect the more efficient removal of LDL and HDL lipoproteins during the plasmapheresis procedure than of lipoproteins containing proportionally more triglyceride (chylomicrons, very low density and intermediate density lipoproteins) as triglyceride concentration was within reference limits after the procedure. The plasma lipid results obtained immediately after the plasmapheresis procedure were not released for reporting. Lipid concentrations had recovered to full plasma concentration levels for the patient more or less by eight days when compared with the steady state concentrations measured at four months post-plasmapheresis. With regard to the liver function tests, GGT (144 to 34 IU/L) and ALT (327 to 37 IU/L) concentrations were reduced to normal and total bilirubin concentrations reduced by 82% (417 to 74 μmol/L) by the procedure. Of interest, ALP concentrations were reduced to subnormal concentrations and did not recover to within reference limits until eight days after the procedure. ALT concentrations subsequently increased, reaching a peak at seven days post the procedure and returning to within reference limits after a further eleven days. It is postulated that the differences seen in the rate of the return of plasma enzyme concentration to steady state concentration may be related to their differing extravascular distribution and the effect that residual disease activity (red cell destruction) may have had on their concentrations. Of the individual proteins analysed after the plasma exchange, plasma albumin and fibrinogen concentrations (given intravenously on the second day post-plasmapheresis) were normal as a result of administration. All other plasma proteins analysed were low in concentration, with levels increasing to steady state concentrations by eight days post-plasmapheresis except for caeruloplasmin, which required a longer time (within reference limits when analysed the next time at four months post-plasmapheresis). Plasma immunoglobulins increased in concentration up to eight days, but still remained low at four months; hypogammaglobulaemia is a common feature in patients with Evans syndrome. 16
This case report illustrates the difficulties of obtaining accurate information on the steady state concentrations of plasma analytes when analysis is carried out on a blood sample from a patient that has recently undergone plasmapheresis. The normal plasma albumin in this situation did not flag the possibility of the sample being artefactually diluted. It is also an example of the importance of supplying clinical details and/or details of relevant treatments when requesting tests. From the data presented here, it would appear that the plasma concentration of some proteins may take longer than eight days to reach steady state concentrations post-plasmapheresis (four daily plasma volume exchanges in this case). This has the potential to produce misleading results if analysis is carried out soon after plasma depletion has occurred, not only in situations where analysis of individual plasma proteins is undertaken, but also in the assessment of the status of hormones and other substances that are significantly bound to plasma proteins other than albumin.