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Fibrinogen levels in Polycythemia Vera patients and hematocrit target for performing a therapeutic phlebotomy in southwest Alabama.

ABSTRACT

Patients with Polycythemia Vera undergo therapeutic phlebotomies on a routine basis to decrease their hematocrit. The blood collected is discarded and could potentially be used in other capacities such as research or controls in the clinical laboratory. In this study the patients' plasma was tested for the coagulation protein fibrinogen to ascertain adequate levels with possible uses of the plasma in the laboratory. In coagulation testing it is advocated to correct the anticoagulant volume in patients with hematocrits above 55% so that residual sodium citrate in the collection tube does not neutralize calcium in the test system and yield erroneous results. Because this step applies only to hematocrits above 55% the target hematocrit for performing a therapeutic phlebotomy was also analyzed. Results demonstrated that nine out of ten patients exhibited adequate levels of fibrinogen and only one patient had a hematocrit above 55% With the majority of clinicians performing therapeutic phlebotomies well below a hematocrit of 55%, it seems unlikely that a correction of sodium citrate is performed on a routine basis in coagulation testing. Plasma, which is routinely discarded, could potentially be used in many other facets of medicine.

INTRODUCTION

Polycythemia vera (PV) is a clonal stem cell disorder characterized by excessive proliferation of red blood cells with possible increases in white blood cells and platelets. The median age of diagnosis is 60 yrs, and signs and symptoms include: hematocrit level above 52% for men and above 48% for women, plethora, pruritus after bathing, splenomegaly, weight loss, and weakness. Patients are also at a risk of thrombosis and transition to acute leukemia. Without treatment, the median survival in untreated symptomatic patients is 6 to 18 months, and with treatment the median survival is more than 10 yrs. (Berk et al., 1986). This disorder was first described by Vaquez in 1892 (Vaquez., 1892). The most common form of treatment for these patients in reducing the red cell mass is a therapeutic phlebotomy (TP) in which approximately 500 mL of blood is withdrawn by venesection on a periodic basis using varying post-TP target hematocrits.

Another disorder which requires TP is hereditary hematochromatosis (HH). Individuals with HH absorb excessive amounts of iron and must undergo periodic TPs to prevent iron accumulation to toxic levels throughout the body. Currently, the Food and Drug Administration provides special variances for blood centers to use a unit of blood from patients with HH for allogeneic use once that unit has undergone the required testing (Brittenham et al., 2001). The basis for this research was to ascertain the level of fibrinogen in PV patients who have undergone a TP and the possible use of salvaged plasma in research or as controls in coagulation testing. Currently, once the unit is drawn from the PV patient, it is discarded. In patients with hematocrit levels exceeding 55%, the volume of anticoagulant (sodium citrate) should be corrected when testing for coagulants such as fibrinogen. The formula is as follows: x = (100-Hct)/(595-Hct) x vol, where x is the volume of anticoagulant required for proper anticoagulation of blood, Hct = patient's hematocrit, and vol = volume of blood to be anticoagulated. The basis for this correction is when there is a high red cell volume, there is a concomitant low plasma volume in the test sample, with subsequent reduced calcium levels. The anticoagulant sodium citrate prevents clotting by binding calcium; if there is less calcium present in the patient's plasma, residual sodium citrate in the test sample will bind calcium used in the test assay, leading to erroneous results. The need for this correction based on target hematocrits was also addressed in this study.

MATERIALS AND METHODS

This study was approved by the Institutional Review Board of Auburn University Montgomery. The subject population consisted of PV patients undergoing a TP at Evergreen Medical Center (EMC). Patients with a history of afibrinogenemia, dysfibrinogenemia, or hypofibrinogenemia were excluded from this study. Informed consent was ascertained by the staff of EMC laboratory. The hematocrit was determined prior to patients'undergoing the TP as ordered by their physician. The hematocrit was performed on the Coulter MAX-M hematology analyzer. The venipuncture site was identified by the EMC medical technologist and scrubbed with alcohol for at least 30 s in all directions from the site of inteded venipuncture. A tourniquet was applied to the patient's arm to increase distention of the vein, and a 21-gauge vacutainer needle was inserted into the patient's vein. The opposite end of the needle or rubber sheath was placed into a 5 mL vacutainer tube containing 0.45 mL of 3.2% sodium citrate, and the tube was labeled with a numerical identifier. The ratio of whole blood to anticoagulant (sodium citrate) was 9:1. The rubber sheath was then placed into a 450 mL glass vacutainer bottle to complete the TP. The sodium citrate vacutainer tube was centrifuged at 2500 x g for 15 min to separate the plasma from the red cells. If the patient's hematocrit exceeded 55% before TP, the sodium citrate volume was corrected using the formula above. The technologist at EMC transferred the plasma to a plastic tube, which was then frozen at-30[degrees]C. Within 24 h the specimen was transported on ice to Auburn University Montgomery for fibrinogen analysis.

The instrument used for analysis was the fibrometer (Becton-Dickinson, New Jersey), which is an electro-mechanical instrument that detects the presence of a fibrin clot via a stationary electrode and a moving electrode. In this assay, the strong enzyme thrombin cleaves the molecule fibrinogen, leaving a fibrin clot. When the clot completes an electrical circuit between electrodes the timer will stop and the time for clot formation will be displayed in seconds. A standard curve was made using reference plasma containing a known quantity of fibrinogen (307 mg/dL.) Dilutions of 1:5, 1:10, 1:20, and 1:40 were prepared using reference material and Imidazole buffered saline (IBS) at pH 7.4. A normal control and patient plasma were diluted 1:10 with IBS. 0.2 mL of each dilution was placed in a plastic fibrotube and warmed to 37[degrees]C for 4-6 min. 0.1 mL of bovine thrombin reagent was added to each dilution tube, and the clotting time was determined on the fibrometer. All tests were run in duplicate.

The standard curve was made by plotting the average of the two clotting times for dilutions of the reference plasma onto semi-log graph paper, where time in seconds on the y-axis is plotted against fibrinogen concentration on the x-axis. For the 1:5 dilution, the fibrinogen concentration is multiplied by 2; for the 1:10 dilution, the fibrinogen concentration is multiplied by 1; for the 1:20 dilution, the fibrinogen concentration is multiplied by 0.5; and for the 1:40 dilution, the fibrinogen concentration is multiplied by 0.25. The values were plotted against the clotting time in seconds, and the best fit line was drawn. Plasma diluted 1:10 represents 100% of the assigned value. To determine the fibrinogen concentration of the normal control and that of patients, the clotting time was located on the y-axis, and the corresponding fibrinogen level on the x-axis was identified. If the clotting times for the control or patients fell outside of the linear curve, 1:5 or 1:20 dilutions were prepared. In the former, the value would be divided by 2; in the latter, the value would be multiplied by 2.

For hematocrit determination, a 7-mL vacutainer tube containing liquid potassium EDTA (ethylene diamine tetraacetic acid) was drawn from each patient before TP. The sample was aspirated into the Coulter MAX-M, and the hematocrit was determined by the calculation RBC x MCV/10, where RBC is red blood cell count and MCV is mean corpuscular volume.

RESULTS

The patient hematocrit values ranged from 41.4 to 55.9%. The fibrinogen levels ranged from 90 to 580 mg/dL and are depicted in Fig. 1. None of the values fell outside of the linear curve, and duplicate runs were within 10%. None of the plasma samples were hemolyzed or lipemic.

The correlation coefficient, r, was -0.3, indicating there was no relationship between the variables hematocrit and fibrinogen. With the exception of one patient, all fibrinogen levels were within normal range (200 to 400 mg/dL) or above normal. Only one patient had a hematocrit above 55% in which correction of the anticoagulant was necessary.

DISCUSSION

The Polycythemia Vera Study Group (PVSG) was organized in 1967 to identify the optimal approach to the diagnosis and treatment of PV (Streiff et al., 2002). In a survey conducted in 2000 by PVSG, investigators found that of the physicians who treat PV with TP, most respondents of the survey used a target hematocrit of 44% or less for phlebotomy therapy. Approximately 24% of physicians used a hematocrit target of 42%, 61% used a target of 44%, 15% used a target of 50%, and only 2% used a target of 55% (Streiff et al., 2002). Although an initial target hematocrit of 52% for TP was originally set by the PVSG (Berk et al., 1981), recent literature suggests the PVSG has discontinued using the target hematocrit number and has instead focused on a hematocrit value post-treatment: maintain the hematocrit at less than 45% (Stuart et al., 2004). This is due in large part to an increased risk of thromboses with high hematocrits (Solberg et al., 2002). It was clear from the patients enrolled in this study that symptoms such as excessive itching and abdominal discomfort occurred at much lower hematocrits than 52%, prompting a physician's order for a TP.

[FIGURE 1 OMITTED]

Other therapies for treating PV patients include myelosuppressive agents such as chlorambucil, radioactive phosphorus ([.sup.32]P), hydroxyurea, and interferon-gamma. Plateletpheresis may also be used to reduce a markedly elevated platelet count. The only cure for PV is a bone marrow transplant.

In this study, nine out of ten patients exhibited adequate levels of fibrinogen in their plasma. The patient with the low fibrinogen level had a hematocrit above 55% with a concomitant lower plasma concentration of fibrinogen. With the majority of physicians ordering TP's at hematocrits less than 55%, it seems unlikely that the anticoagulant volume is corrected on a routine basis. Plasma from a TP is routinely discarded and could be used in a clinical laboratory setting as controls in coagulation testing or in proficiency testing, or further processed to make buffy coats (concentrated white blood cells and platelets) or a variety of plasma products (i.e. cryoprecipitate) to be used in research. Unlike the scenario with HH patients, this blood could not be used for in vivo use or transfusion to other patients as PV is a form of cancer and can transform into leukemia. With a PV incidence of 2.3 per 100 000 in the general population and TP as the most common treatment, blood that is discarded from this procedure could be used in many other facets of medicine.

LITERATURE CITED

Berk, P.D., J.D. Goldberg, M.N. Silverstein, A. Weinfeld, P.B. Donovan, J.T. Ellis, S.A. Landaw, J. Laszlo, Y. Najean, A. V. Pisciotta, and L.R. Wasserman. 1981. Increased incidence of acute leukemia in polycythemia vera associated with chlorambucil therapy. New England Journal of Medicine 304: 441-447.

Brittenham, G.M., H.G. Klein, J.P. Kushner, and R.S. Ajioka. 2001. Preserving the national blood supply. Hematology 1: 422-430.

Solberg, L.A. 2002. Therapeutic options for essential thrombocythemia and polycythemia vera. Seminars in Oncology 29(3 Suppl 10): 10-15.

Spivak J.L., G. Baroki, G. Tognoni, T. Barbui, G. Finazzi, R. Marchioli, and M. Marchetti. 2003. Chronic myeloproliferative disorders. Hematology (American Society of Hematology Education Program) 200-24.

Streiff, M.B., B. Smith, J.L. Spivak. 2002. The diagnosis and management of polycythemia vera in the era since the polycythemia vera study group: a survey of American Society of Hematology members' practice pattern. Blood 99: 1144-1149.

Stuart, B.J., and Viera, A.J. 2004. Polycythemia vera. American Family Physician 69: 2139-44.

Vaquez, J.M. 1892. Sur une forme speciale de cyanose s'accompagnant d' hyperglobulie excessive ET peristante. Social Biology (Paris) 44: 384-388.

Virginia C. Hughes*

Alice L. Anderson

Doris C. Davidson

Auburn University Montgomery

Montgomery, AL 36124-4023

*Contact author
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Author:Hughes, Virginia C.; Anderson, Alice L.; Davidson, Doris C.
Publication:Journal of the Alabama Academy of Science
Geographic Code:1USA
Date:Jan 1, 2006
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