Answering your questions

Q

Occasionally, we hear complaints from pediatricians that we report too many bands in our manual differentials. Our laboratory adheres to the CAP classification criteria that require seeing a filament in order to call a seg, a seg. If the nucleus is folded on itself and it’s hard to see what’s underneath, we go with the more mature cell.

Our medical director has asked us to modify our segs and bands criteria so that we call fewer bands. This request, although triggered by a physician inquiry, is primarily based on our performance on New York State Cytohematology proficiency testing, where we regularly fall in the upper end of the acceptable range for bands.

I have not been able to find a reference for classifying segs and bands that is not based on the CAP criteria. In order to retrain our techs it would be very helpful to cite a reputable source. Any assistance with this request would be greatly appreciated.

A

Neutrophils dominate the normal adult peripheral blood and may be found in two forms: segmented neutrophils (1,800-7,800/µL) and band neutrophils (0-700/µL).¹ Though traditionally counted as two separate categories, there is growing controversy as to their utility in clinical patient management.²

The College of American Pathologists defines a band neutrophil (stab or staff cell) as follows: “Bands are the same size or slightly smaller than metamyelocytes. The nucleus is centrally or eccentrically placed and indented to more than half the distance from the farthest nuclear margin. The nucleus may appear in the shape of a band, sausage, letters C or U, or may be lobulated. If lobulated, the bridge or isthmus between the lobes must be wide enough to have two distinct parallel dark margins with light nuclear material in between.”³

Other sources define bands in a similar manner.^2,4-6 And yet others offer a more relaxed definition: “a constriction greater than one half to two-thirds of the nuclear breadth as adequate evidence of lobulation and classify such cells as polymorphoneuclear.”⁷ 

Regardless of the definition or how bands have traditionally been classified, current thinking is that “a band count is a nonspecific, inaccurate, and imprecise laboratory test.”² Furthermore, in a recommendation, the Clinical and Laboratory Standards Institute (CLSI) states that “the inclusion of ‘bands’ as a separate category of leukocyte is not supported by medical data.”⁸

The CAP Hematology and Clinical Microscopy Resource Committee does not support the idea of using band counts as part of patient management. Yet, band counts in infants under the age of three months are still often requested by pediatricians and neonatologists when dealing with a risk of infection.⁹ 

Reference intervals can vary greatly due to inter- and intra-laboratory variations and the statistical limitations related to cell count sampling (100-200 cells). Though increased presence of band neutrophils may be seen in a variety of diseases such as bacterial and viral infections, inflammatory processes, neoplasms, and other diseases, the WBC and the absolute neutrophil count may serve as a better diagnostic tool.^2,10,11 It should also be noted that when it is uncertain whether a cell is a segmented neutrophil or a band, it is recommended that it be assigned to the former classification.⁶ 

References

1. Henry’s Clinical Diagnosis and Management by Laboratory Methods, 22nd. Saunders Elsevier: Philadelphia. (2011) Appendix A5-9. 1501-1503. 
2. Cornbleet PJ. Clinical utility of the band count. Clinics in Lab Med.2002;22(1):101-136. 
3. Glassy EF. Granulocytic (myeloid) cells. Color Atlas of Hematology: An illustrated field guide based on proficiency testing. Northfield, IL: College of American Pathologists. 1998.22-26.
4. Mathur SC, Schexneider KI, Hutchison RE. In Henry’s Clinical Diagnosis and Management by Laboratory Methods, 22nd. Saunders Elsevier: Philadelphia. 2011. Chapter 31. 536-556. 
5. Glasser L, Fiederlein RL. Functional differentiation of normal human neutrophils. Blood.1987;69:937-944. 
6. NCCLS H20-A. Reference Leukocyte Differential count (proportional) and evaluation of instrumental methods. NCCLS 12(1):1-60 (March 1992) Note: name change to Clinical and Laboratory Standards Institute (2005).
7. Greer JP, Foerster J, Rodgers GM, Skubitz KM. Neutrophilic leukocytes. Clinical Hematology, Vol.1, 12th ed. Lippencott-Williams:Philadelphia. Chapter 9. 2009;170-213.
8. Koepke JA, et al. Clinical Laboratory and Standards Institute. Reference leukocyte (WBC) differential count (Proportional) and evaluation of instrumental methods, 2nd ed. 2007;H20-A2; 27(4).
9. College of American Pathologists. To and fro on band count reporting and clinical utility. CAP Today. 2010(11).
10. Clarke G. Toward abandoning the band—a practice recommendation of the CCQLM. Canadian Coalition for Quality in Laboratory Medicine.2006(5). 
11. http://www.cpsa.ab.ca/Libraries/pro_qofc_alqep_critiques/CCQLM_Band.pdf?sfvrsn=0. Accessed August 10, 2014.
12. Al-Gwaiz LA, Babay HH. The diagnostic value of absolute neutrophils count, band count and morphologic changes of neutrophils in predicting bacterial infections. Med Princ Pract. 2007;16:344-347. 

Q

When a spun urine sediment shows packed WBCs, what procedure is used to quantitate the other elements found in the urine such as bacteria, epithelials, RBCs, etc? Is diluting with saline acceptable? Is there a reference source for this procedure?

A

Urine microscopy has been available as a valuable diagnostic tool since the early 1800s.¹ Best practice in performing a urinalysis requires fresh urine; less than two hours from collection with refrigeration preserving some urinary elements for longer periods, though amorphous and/or crystalline materials may form. Hypotonic or alkaline urine can cause lysis of any WBCs present.² 

Basic urinalysis is performed with reagent strips testing for physiochemical properties of the urine as well as other elements. Based on a laboratory’s established protocol, positive urines may be examined by centrifuging 12mL of well-mixed urine at 400xg for five minutes in a refrigerated (4°C) centrifuge.⁴ The supernatant is aspirated (not decanted) and the sediment resuspended in 250µL of native urine of which a drop is then examined under the micrscope.⁴ For quantitative evaluation, a hemocytometer may be used.^4,5 Use of fixed urine volumes is essential for calculating accurate particle numbers. Manual evaluation of urine sediment may be further enhanced by using supravital stains such as crystal violet/safranin O stain and phase miscroscopy.²

For concentrated urines, uncentrifuged specimens may be examined. The literature does not specifically address the dilution of urine samples. The presence of one WBC per low-powered field (10X) in an uncentrifuged sample approximates 3X10³ WBCs/mL in urine. The presence of 30 WBCs/low powered field suggests that the urine contains >10⁵ WBCs per mL of urine and is consistent with a urinary tract infection.⁵ 

Advances in urinalysis technology have offered greater sensitivity, reproducibility, and accuracy in addition to reducing time-consuming manual preparation and examination of urine sediments. Several technologies have been introduced in the automation of urinalysis. Automated reagent strip readers can standardize the interpretation of color changes using reflectance photometry and are commonly used in many laboratories.⁴ 

In addition, several fully automated urinalysis systems are in current use.^6-9 One type uses video stop-motion camera technology to capture hundreds of frames of digital images of cells, yeast, bacteria, casts, and crystals. These images can be stored and reviewed accordingly to determine the number and type of particle present. 

In another system, flow cytometry technology has been used. As urinary elements pass through a laminar flow cell, laser illumination provides forward angle and side scattering events that are used to identify and quantitate white and red cells, bacteria, epithelial cells, and casts, in addition identifying the presence of yeast, crystals, dysmorphic RBCs, and abnormal casts.^6,7 Because fixed urine sample sizes are used, highly accurate evaluations by both methods can provide clinical information previously not available. Knowing the numbers of cells and bacteria present is especially useful when following serial specimens of patients undergoing treatment.²

It should be noted that when centrifuged, samples may lose certain elements, especially RBCs and WBCs.³ Consistency in preparing urine sediments—fixed urine volume to be centrifuged, centrifuge speed, centrifuge tube size, and proper aspiration of the supernatant—all  contribute to variability in sediment evaluation. Fully automated urinalysis systems offer a faster, accurate, and more consistent way to manage a high volume test with minimal technical assistance.

References

1. Fogazzi GB, Garigali G. The clinical art and science of urine microscopy. Current Opin in Nephrolog Hyperten.2003;12:625-632.
2. McPherson RA, Ben-Ezra J. Basic examination of urine. In Henry’s Clinical Diagnosis and Management by Laboratory Methods, 22nd. Elsever-Saunders:Philadephia.2011. Chapter 28.445-479.
3. European Confederation of Laboratory Medicine. European urinalysis guidelines. Scand J Clin Lab Invest. 2000;60:1-96. 
4. Block DR, Lieske JC. Automated urinalysis in the clinical lab. MLO. 2012; 44(10):8-12.
5. Musher DM, Thorsteinsson SB, Airola VM. Quantitative urinalysis: Diagnosing urinary tract infection in men. J Amer Med Assoc.1976;226(18):2069-2072.
6. Ben-Ezra J, Bork L, McPherson RA. Evaluation of the Sysmex UF-100 automated urinalysis analyzer. Clin Chem. 1998;44(1):92-95.
7. Ottiger C, Huber AR. Quantitative urine particle analysis: Integrative approach for the optimal combination of automation with UF-100 and microscopic review with KOVA cell chamber. Clin Chem. 2003;49(4):617-623.
8. Wah DT, Wises PK, Butch AW. Analytic performance of the iQ200 automated urine microscopy analyzer and comparison with manual counts using Fuchs-Rosenthal cell chambers. Am J Clin Pathol. 2005;123:290-296.
9. Chen YL, Chang MK, Chen YJ, Chang BC. Comparing Neubauer hemacytometer, SY conventional, SY located, and automated flow cytometer F-100 methods for urinalysis. Lab Med. 2009;40(4):227-231.