Lipemia and hyperleukocytosis can lead to CBC errors

This is a follow-up question to “What CBC parameters are affected when the specimen is lipemic?” printed in March 2016, issue [MLO.2016;48(3):44].

Q How should we correct for the hemoglobin and other RBC indices? We’ve been using “hemoglobin blank” or “saline replacement” to do this. However, I could not find the resources/references about these methods. Also what is your preference: “hemoglobin blank” or “saline replacement”?

In addition, do you have any references on how to correct other CBC parameters if the WBC count is more than 300,000/uL?

A Blood specimens that have endogenous interfering substances, such as bilirubin, hemoglobin, paraproteins, and lipids, can interfere with obtaining accurate laboratory results.¹ When triglycerides exceed 300 mg/dL, turbidity occurs, directly affecting the ability to accurately obtain certain CBC parameters.² Lipemia has been reported as a source of interference when measuring hemoglobin, white blood cells,⁴ platelets,⁵ and even lymphocytes.⁶

Lipemia and the CBC

When a patient’s MCHC value is 36 g/dL or greater, it would strongly suggest the presence of an interfering substance. Lipemic specimens contain high levels of triglycerides consisting of chylomicrons and very low-density lipoprotein particles, which in turn cause turbidity. This turbidity interferes with light scatter and the absorption of light, resulting in a false increase of hemoglobin determinations.

When it is determined that a patient sample is lipemic, notifying the ordering practitioner is important in order to determine if a patient can be put on a lipid-free diet with follow-up blood work a few hours later.⁷ If it is critical to obtain CBC results, a point-of-care, whole-blood analyzer may be appropriate. Another approach is to use a saline replacement technique. An EDTA-sample is centrifuged and plasma carefully removed without disturbing the buffy coat. An equal volume of normal saline replaces the discarded plasma and  the tube is mixed, and processed for CBC evaluation. Reporting of results should include notation of lipemia and how the specimen processing was modified.

Zeng et al. employed a correction formula that appears to be fairly reliable.³ The lipemic whole blood sample is run an analyzer (LB) to obtain the Hct (LB) and RBC(LB). A portion of the whole blood is centrifuged (550g x 3 mins) and a manual hemoglobin determination is obtained k(LP) on the plasma. The
following formula can then be used:

• Hgb_(Corrected) = Hgb_LB – (Hgb_LP – Hgb_LP X Hct_LB)

RBC indices may then be calculated as:

• MCH_(Corrected) = Hgb_(Corrected)/RBC_(LB)
• MCHC_(Corrected) = Hgb_(Corrected)/Hct_(LB)

Hyperleukocytosis and CBC

Leukocytosis occurs when the white blood cell count is generally greater than 10,000-11,000/µL, while hyperleukocytosis is when the WBC count is greater than 100,000/µL. Hyperleukocytosis has been reported to occur between five percent and 13 percent in AML, 10 percent to 30 percent in ALL, and rarely in CLL. While counts greater than 100,000 cells/ µL do occur, the highest reported WBC is 1,052,000/µL.⁸ The presence of hyperleukocytosis is a poor prognostic indicator because most patients incur leukostasis (vascular obstruction/tissue hypoxia), tumor lysis syndrome, and/or disseminated intravascular coagulation (DIC).⁹

Hyperleukocytosis can affect the accuracy of platelet, hemoglobin, and even MCV determinations.⁸ Today’s hematology analyzers are quite robust, and most are able to detect unlikely results that “flag” the operator, indicating careful review of the results is needed. Establishing WBC linearity is important to determine the upper limit of reliability. In one study, WBC linearity was shown to range from zero to 400,000/µL.¹⁰ Thus, appropriate dilutions of a well-mixed EDTA specimen with an isotonic solution can generally render an accurate WBC count. Each laboratory should determine the linearity of its respective hematology analyzer.

Hematocrits on hyperleukocytic specimens can be obtained through a standard capillary, microcentrifuge technique. One must not include the leukocrit (buffy coat section of the spun hematocrit tube) as part of the hematocrit determination. Noting what the leukocrit is can be of value in evaluating the WBC mass (tumor burden). Pre- and post-WBC counts may be performed and leukocrits may be used in determining effects of treatment through chemotherapy or leukophoresis.⁹

Hemoglobin, RBC, and MCV determinations are more difficult to evaluate when such elevated white cell counts are encountered. Due to the WBC turbidity, direct hemoglobin values may not be reliable. The high number of WBCs may be included in the RBC count, particularly with analyzers utilizing impedance measurements; thus, WBC values can be subtracted from the RBC. In a similar fashion, MCV determinations may include a large number of white cells, many of which are large, leukemic cells, and may not produce reliable results.

Conclusion

Each laboratory must establish the appropriate “interference” protocols that best address the technical skills, equipment available, and clinician approval. The protocols should include references to proper pre-analytic procedures (minimize lipemia by fasting eight to 12 hours before blood draw, particularly when dealing with outpatients), how lipemic specimens are to be identified, the procedure on how lipemic specimens will be handled, and how to ensure appropriate information is presented to the healthcare practitioner.

In a similar fashion, a protocol should also be established for handling hyperleukocytic specimens with appropriate references to manual CBC methods. Completing linearity studies are critical in establishing such a protocol. Regardless of what the interfering substance is, prompt physician notification is essential to ensure timely patient management.

REFERENCES

1. Kroll MH, Elin RJ. Interference with clinical laboratory analyses. Clin Chem. 1994;40(11):1996-2005.
2. Roche Diagnostics. Serum indices: Reduction of clinical errors in laboratory medicine. 2007. www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=14&ved=0ahUKEwiGrdK1vZPKAhWDOT4KHVD3ANA4ChAWCCowAw&url=http%3A%2F%2Froche-diagnostics.cz%2FProducts%2FDocuments%2FPD%2FSpolecne%2FSerove_indexy.pdf&usg=AFQjCNFxgWHNhCsuzIGAjxkTislu5eVJFQ&sig2=z2TkOVU1IGgM0bO0W8K1_w (Last  accessed 1/4/2016).
3. Zeng SG, Zeng TT, Jiang H, et al. A simple, fast correction method of triglyceride interference in blood hemoglobin automated measurement. J Clin Lab Analysis. 2013;27:341-345.
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6. Er TK, Gines MAR, Chen YT, et al. Erroneous result of white blood cell differential count in a patient with mixed hyperlipidemia. Clin Chem Lab Med. 2008;46(7):1054-1055.
7. Savage RA. CAP Today. July 2004. http://www.captodayonline.com/Archives/q_and_a/qa_07_04.html.
8. Berz D, Freeman NJ. Extreme lymphocytosis. J Clin Oncolog. 2008;26(4):674-679.
9. Ganzel C, Becker J, Mintz PD, et al. Hyperleukocytosis, leukocytosis and leukapharesis: Practice management. Blood Rev. 2012;26:117-122.
10. Walters J, Garrity P. Performance evaluation of the Sysmex XE-2100 Hematology analyzer. Lab Hematol. 2000;6:83-92.