Q & A

December 2004

Richard A. Savage, MD, Editor

Q.  What are the correct guidelines for triaging a pediatric cerebrospinal fluid three-tube specimen sent for hematological cell count, chemical analysis for total protein, and microbiological culture? In Tietz’s Textbook of Clinical Chemistry, 3rd edition, the authors state that examination of CSF total protein is chiefly used to detect increased permeability of the blood-CSF barrier to plasma protein and that blood in the CSF specimen invalidates the CSF protein values. Does this imply that a valid CSF total protein value may be realized only when the same tube of CSF specimen is used for both the total protein and cell count?

A.  When triaging a three-tube pediatric cerebrospinal fluid specimen, the first tube should be used for chemistry and immunology studies, the second tube for microbiologic studies, and the third tube for cell count and differential. It is not necessary to perform the chemistry, cell count, and differential on the same tube to achieve valid results. The biggest concern is to perform the cell count and differential on the clearest tube received, which is generally the third tube. The specimen should be sent to the laboratory immediately and processed quickly to minimize cellular degradation.

Member, CAP Hematology/Clinical
Microscopy Resource Committee

Q.  What battery of tests is recommended for evaluating a patient for possible spontaneous deep vein thrombosis—for instance, the DVT patient who is not postoperative or postpartum? Of these tests, which need to be drawn in the emergency department before initiating anti-clot therapy?

A.  To test for spontaneous, or unprovoked, venous thrombosis, the recommended tests include factor V Leiden (an activated protein C resistance assay with factor V deficient plasma can be used to screen, with confirmatory DNA testing if the screen is abnormal), functional protein C, functional protein S, functional antithrombin, and prothrombin G20210A. Anticardiolipin antibody and lupus anticoagulant tests are also appropriate, especially if there is no family history of thrombosis. Although somewhat controversial, homocysteine testing can also be considered because elevated levels can be lowered with vitamins B₁₂, folate, and B₆.^1,2

It is worthwhile to test for protein C, protein S, antithrombin, and lupus anticoagulants before initiating anticoagulant therapy. Warfarin decreases levels of protein C and protein S. Heparin can cause slight decreases in antithrombin III. If heparin is present, the laboratory should be notified before performing lupus anticoagulant tests because heparin can interfere with certain assays. Some coagulation laboratories routinely perform procedures to detect heparin, while others do so only on request.

In addition to protein C, protein S, antithrombin, and lupus anticoagulants, it is worthwhile to test for activated protein C resistance prior to initiating the new anticoagulants argatroban or hirudin. Argatroban and hirudin are useful for patients who cannot tolerate heparin, such as those with heparin-induced thrombocytopenia. These two anticoagulants inhibit thrombin and, therefore, interfere with most assays that involve a clotting time. This includes false elevations or no-clot results with clot-based protein C or protein S assays and false-positive lupus anticoagulant assays. Thrombin-based chromogenic antithrombin assays will be falsely elevated because the assay detects thrombin inhibition. It is not yet known what effect argatroban or hirudin have on the activated protein C resistance assay.

References

1. Van Cott EM, Laposata M, Prins MH. Laboratory evaluation of hypercoagulability with venous or arterial thrombosis. College of American Pathologists Consensus Conference XXXVI. Arch Pathol Lab Med. 2002;126:1281-1295.
2. Key NS, McGlennen RC. Hyperhomocyst(e)inemia and thrombophilia. College of American Pathologists Consensus Conference XXXVI. Arch Pathol Lab Med. 2002;126:1367-1375.

Member, CAP Coagulation Resource Committee

Q.  Which deep vein thrombosis tests can be obtained within a day or two of presentation to the ER, while on anticoagulant therapy, and during the clotting episode? On the flip side, which tests require the patient to be off anticoagulant therapy and not actively clotting and, thus, will have to be obtained three to six months after the patient initially presents to the emergency department? Is fasting recommended?

A.  The activated protein C resistance assay with factor V deficient plasma is typically not affected by heparin—as long as the assay contains a heparin neutralizer—or warfarin or current thrombosis. DNA tests, such as factor V Leiden and prothrombin G20210A, are not affected by anticoagulants or current thrombosis, nor are homocysteine and immunoassays, such as anticardiolipin antibody assays.

Protein C, protein S, and antithrombin III become consumed during thrombosis, and their levels may be decreased during an acute episode. However, in my experience, with most patients these three proteins remain within the normal range despite acute thrombosis. Therefore, I recommend that these assays be performed at the time of presentation, before initiating anticoagulation. If the result is normal, it can be inferred that the patient does not have a hereditary deficiency of one of these three proteins. If antithrombin III is decreased, retesting anytime when the patient has not received heparin for at least five days would be needed before establishing a diagnosis.¹ If the result for protein C or protein S is decreased, retesting anytime when the patient has not received warfarin for at least 10 days would be needed before establishing a diagnosis. In fact, a delay of 30 days after discontinuing warfarin has been recommended for protein S,² because this protein takes longer to return to normal than protein C following warfarin discontinuation. Testing first-degree relatives, or any relative with a history of venous thrombosis, can also be informative because hereditary deficiencies of protein C, protein S, or antithrombin have autosomal dominant inheritance.

Homocysteine is the only test in the recommended panel for which fasting is a consideration. In some reports, homocysteine is slightly elevated by food consumption, while in other reports, homocysteine is slightly decreased or unchanged.³ Thus, it is possible that the most reliable homocysteine assessments are obtained with fasting specimens. However, due to the small and inconsistent effects of food consumption on homocysteine and the impractical nature of requiring fasting specimens for hypercoagulation testing, many laboratories do not require fasting specimens.

College of American Pathologists Consensus Conference XXXVI on thrombophilia testing did not make fasting a recommendation because “it is unclear whether it is necessary to insist on fasting specimens” for homocysteine testing.⁴ It is more important to keep the specimen on ice after collection and to immediately transport the specimen to the laboratory for processing because red blood cells continue to produce and release homocysteine in the specimen after collection, causing spurious elevations in homocysteine if specimen processing is delayed.

For further details regarding evaluating patients with DVT, refer to the November 2002 issue of Archives of Pathology & Laboratory Medicine, which is devoted to diagnostic issues in thrombophilia.

References

1. Kottke-Marchant K, Duncan A. Antithrombin deficiency: issues in laboratory diagnosis. College of American Pathologists Consensus Conference XXXVI. Arch Pathol Lab Med. 2002;126: 1326–1336.
2. Goodwin AJ, Rosendaal FR, Kottke-Marchant K, et al. A review of the technical, diagnostic, and epidemiologic considerations for protein S assays. College of American Pathologists Consensus Conference XXXVI. Arch Pathol Lab Med. 2002;126:1349–1366.
3. Ueland PM, Refsum H, Stabler SP, et al. Total homocysteine in plasma or serum: methods and clinical applications. Clin Chem. 1993;39:1764–1779.
4. Key NS, McGlennen RC. Hyperhomocyst(e)inemia and thrombophilia. College of American Pathologists Consensus Conference XXXVI. Arch Pathol Lab Med. 2002;126:1367–1375.

Member, CAP Coagulation Resource Committee

Q. Our official procedure for a reticulocyte count is the Miller disk method. Some employees, however, use a method in which they count about 200 red blood cells in a field and then count the retics in that field and four other fields that appear to have 200 RBCs each and say it is equal to 1,000 RBCs. Is this accurate and acceptable?

A. What these employees are doing is similar in principle to the Miller disk. Both count representative areas to decrease the total number of RBCs counted. The Miller disk estimates based on counting a portion of each field, whereas your coworkers estimate using a gestalt of what a 200-cell field looks like. The Miller disk has been validated and standardized; your coworkers’ method is based on each individual’s ability to correctly identify comparable fields. This is not, to my knowledge, a recognized method of doing a reticulocyte count, and it would be difficult to validate because it is based in part on each individual's ability to correctly estimate the comparability of fields. This method is too subjective; laboratory testing needs to be based on objective observations.

Robert Novak, MD
Department of Pathology Children's Hospital Medical
Center of Akron (Ohio)

Chair, CAP Hematology/Clinical
Microscopy Resource Committee