Q & A

June 2004

Richard A. Savage, MD, Editor

Q.  We recently implemented a policy not to re-spin blood samples that arrive at our reference laboratory if they arrive in a serum-separator tube. Our policy is to pour them off and re-spin because re-spinning the primary tube would falsely elevate the potassium. Several of our technologists claim that other labs re-spin the primary tubes with no adverse effect. Can you clarify this matter?

A.  Hyperkalemia continues to be a problem for reference laboratories, in large part because blood collection is frequently out of the lab’s control and testing is often delayed when compared to samples collected in a hospital system. Preanalytical causes of hyperkalemia include excessive fist clenching,¹ tight tourniquet,²delay in centrifugation with resultant release of potassium from cellular elements, release of potassium into serum during the clotting process from platelets during thrombocytosis,³ or leukocytes during leukocytosis,⁴ and contamination from EDTA tubes that were collected before the routine chemistry tube.

If serum or plasma is used, hemolysis, which also causes hyperkalemia, should be obvious to lab personnel. However, if whole blood is used and hemolysis is induced by shear forces in the syringe or short catheters,⁵ it may not be detected unless the plasma is prepared for visual inspection. Our laboratory is also dealing with the problem of recentrifuging a serum-separator tube. NCCLS does not recommend recentrifuging.⁶

The literature on recentrifugation is relatively sparse, but there are three reports that may be helpful. Two studies^7,8 describe experiences in large automated laboratories in Japan, where hyperkalemia was noted in several patients whose samples had been recentrifuged. The authors’ investigations confirmed that recentrifugation was the cause of the hyperkalemia, and the policy of recentrifugation was discontinued. Using a volunteer with a baseline potassium of 3.95 mmol/L, the authors found potassium increased following recentrifugation at 24 hours, 48 hours, and 72 hours to 5.95 mmol/L, 6.90 mmol/L, and 7.61 mmol/L, respectively. These increases were not related to hemolysis. One study⁷ also reviewed mean laboratory potassium levels before and after recentrifugation was discontinued. The mean value was 4.68 mmol/L before the change and 4.14 mmol/L afterward. In a 1991 study, Hue et al⁹ reported unexpected hyperkalemia, traced to recentrifugation, in an outpatient. The authors also investigated the time and speed of the initial spin. Their usual centrifugation policy was for 10 minutes at 2,800 ¥ g. If this initial time was increased to 25 minutes, the effect of increasing potassium following recentrifugation was eliminated. However, the increased time for centrifugation was considered impractical for a busy laboratory and perhaps for a busy doctor’s office.

We recently performed in-house studies that support the results of Hira et al^7,8 and Hue et al⁹. One study⁸ investigated recentrifugation at and beyond 24 hours. We wanted to know at what time the recentrifugation began to have a significant effect. Based on a small number of volunteers and using the physician’s office-type centrifuge, we found that by 12 hours after collection recentrifugation led to an increase of 0.6 mmol/L compared to the control samples.

The theory behind increased potassium after recentrifugation is that on initial centrifugation, the cells are separated from the serum by thixotropic gel. When centrifuged, this gel becomes less viscous and, based on the specific gravities of the components of whole blood, positions itself between cells and serum (or plasma in a plasma-separator tube). The general opinion is that a small amount of serum usually persists between the cells in the clot. As the tube sits undisturbed for several hours, potassium leaks out of cells and accumulates in the small amount of remaining serum. The leak is likely to be worse if the tube is refrigerated rather than stored at room temperature.^10,11 When the tube is recentrifuged, the serum remaining with the clot, which now contains a much higher concentration of potassium, is added to the original serum sitting above the gel barrier, leading to a higher potassium level in the combined serum sample.

Busy labs most likely recentrifuge all samples in an attempt to be efficient. Hira et al⁸ reported that their lab received a combination of centrifuged, uncentrifuged, and partially centrifuged tubes and that it required less time to recentrifuge all of the tubes than to identify those that needed recentrifugation. Unfortunately, in an era of increased automation and decreasing staff, this problem may persist and may be why some labs continue to recentrifuge gel-barrier tubes.

The type of centrifuge used may also be related to hyperkalemia. Using a fixed-angle centrifuge results in the gel barrier being on a slant instead of horizontal, as would be the case with a “swinging bucket” centrifuge. Dufour¹² provides a useful description of his experiences with various types of centrifuges. He points out that the gel may not form a complete barrier when a fixed-angle centrifuge is used. Unfortunately, the cost of fixed-angle centrifuges appears to be considerably less than the swinging bucket type, thus potentially perpetuating the problem.

In conclusion, based on our review of the literature and in-house studies, we strongly discourage the recentrifugation of gel-barrier tubes, particularly 12 or more hours after blood collection.

References

1. Don BR, Sebastian A, Cheitlin M, et al. Pseudohyperkalemia caused by fist clenching during phlebotomy. N Engl J Med. 1990;322:1290-1292.
   
2. Skinner SL. A cause of erroneous potassium level. Lancet. 1961;1:478-480.
   
3. Ingram RH Jr, Seki M. Pseudohyperkalemia with thrombocytosis. N Engl J Med. 1962;267: 895-900.
   
4. Bronson WR, DeVita VT, Carbone PP, et al. Pseudohyperkalemia due to release of potassium from white blood cells during clotting. N Engl J Med. 1966;274: 369-375.
   
5. Raisky F, Gauthier C, Marchal A, et al. Haemolyzed samples; responsibility of short catheters. Ann Biol Clin. 1994;52: 523-527.
   
6. Procedures for handling and processing of blood specimens; approved guideline-second edition. NCCLS document H18-A2. 1999;(21):19.
   
7. Hira K, Ohtani Y, Rahman M, et al. Pseudohyperkalaemia caused by re-centrifugation of blood samples after storage in gel separator tubes. Ann Clin Biochem. 2001;38:386-390.
   
8. Hira K, Shimbo T, Fukui T. High serum potassium concentrations after re-centrifugation of stored blood specimens. N Engl J Med. 2000;343:153-154.
   
9. Hue DP, Culank LS, Toase PD, et al. Observed changes in serum potassium concentration following repeat centrifugation of Sarstedt serum gel safety monovettes after storage. Ann Clin Biochem. 1991;28:309-310.
   
10. Oliver TK Jr, Young GA, Bates GD, et al. Factitial hyperkalemia due to icing before analysis. Pediatrics. 1966;38:900-902.
    
11. Ono T, Kitaguchi K, Takehara M, et al. Serum-constituents analyses: Effect of duration and temperature of storage of clotted blood. Clin Chem. 1981;27:35-38.
    
12. Dufour DR. Elevated potassium levels. Medical Laboratory Observer. Feb. 1998: 11-12.

Elizabeth Sykes, MD
Chief, Clinical Chemistry
Department of Clinical Chemistry
Frederick L. Kiechle, MD, PhD
Chairman, Department
of Clinical Pathology
William Beaumont Hospital
Royal Oak, Mich.
Advisor, CAP Publications Committee
Member, CAP EXCEL Advisory Committee

Q.  Should a woman phenotyping as weak D during pregnancy receive Rh immunoglobulin?

A.  This question has no clear-cut answer. First, the laboratory should ensure that the weak D typing is not caused by a massive fetal-maternal hemorrhage.¹ Some would argue that there is no need to administer Rh immunoglobulin to a woman who is phenotypically weak D since the D antigen is not foreign and she would be unable to make an alloantibody against it to threaten future pregnancies. However, a small proportion of weak D women have a partial D phenotype, such as category VI or DVI, and fetal D-positive red cells could alloimmunize her to the portion of the D antigen that she lacks.2 In future pregnancies, this antibody could react as an anti-D. Whether the RhIg would reach any fetal red cells in the maternal circulation is uncertain, however. The maternal cells will be much more numerous and may adsorb most or all the administered anti-D. The appropriate dose of RhIg in this situation is unknown.³ No standardized approach exists for handling maternal weak D situations. Many institutions would phenotype a woman for D early in her pregnancy without using an antiglobulin (weak D) test. If she tests D-negative, she would be a candidate for RhIg. (This approach will result in the unnecessary administration of RhIg to some weak D women, but the virological safety of the RhIg preparation would suggest this to be a low-risk option.) If a weak D test is positive, the medical director of the transfusion service will need to formulate a policy regarding whether to consult obstetrical caregivers before administering RhIg. Most institutions would not administer it.²

References

1. Sebring ES, Polesky HF. Detection of fetal maternal hemorrhage in Rh immune globulin candidates. A rosetting technique using enzyme-treated Rh2Rh2 indicator erythrocytes. Transfusion. 1982; 22:468-471.
   
2. Judd WJ. Practice guidelines for prenatal and perinatal immunohematology revisited. Transfusion. 2001;41: 1445-1452.
   
3. Lubenko A, Contreras M, Habash J. Should anti-Rh immunoglobulin be given to D variant women? Br J Haematol. 1989;72:429-433.
   

James P. Aubuchon, MD
E. Elizabeth French Professor
Chair, Department of Pathology
Dartmouth-Hitchcock
Medical Center
Lebanon, NH
Chair, CAP Transfusion Medicine
Resource Committee