Laboratory 1. CBC Part I: Cells Counts

INTRODUCTION

The minimum performance expectation for this laboratory exercise requires you to:

1. Come to class on time and be prepared to participate by reading this assignment ahead of time!

2. Ask questions.

3. Practice safety.

4. Select and prepare reagents, identify specimens suitable for analysis, and use instruments to generate test data.

5. Apply principles of basic laboratory procedures in order to perform these tests:

ü Manual cell counts

ü Automated cell counts

6. Perform quality control and take corrective actions when necessary to ensure the validity of test results.

7. Plan and select procedural courses of action appropriate for the samples used, the test to be performed, and which allows you to complete tasks accurately, and responsibly in the class time allotted.

8. Evaluate hematology results to identify sources of error in testing and to discriminate between states of health or disease.

9. Calculate results from test data.

10. Assess test and quality control data in order to validate results.

11. Report your results.

12. Develop a hematology lexicon and use the vocabulary to identify, explain, interpret and report data.

IN THE BEGINNING

Hematology is a term which literally means the study of blood. However, practically speaking, hematology involves the evaluation of the blood cells—erythrocytes or red blood cells [rbcs], leukocytes or white blood cells [wbcs], and thrombocytes or platelets [plts]—in terms of the number of cells present in a volume of whole blood, and a descriptive report of their morphology and functionality. There are a number of specific hematology tests which can be done to evaluate a person’s blood. One such test is the complete blood count (CBC). When a doctor orders this test, the report received will include at a minimum the following data:

ü Total white blood cell count [WBC]

ü Total red cell count [RBC]

ü Absolute quantitative hemoglobin [HGB or HB]

ü Hematocrit [HCT]

ü White cell differential

ü Red cell indices [MCV, MCH, MCHC]

CBC data is often used to evaluate a person’s ability to fight disease, to monitor the effect(s) of drug therapy, to formulate a prognosis, and it is used in the diagnosis of disease. While the CBC is one of the most frequently ordered lab tests, physicians do have the option of ordering individual tests [e.g., HCT, or HGB], or combinations of tests that best meet the need of the doctor and patient.

BLOOD CELL COUNTS

Blood cell counts may be performed manually or by using automated cell counters. Manual counts are often performed it cases of extremely low (-cytopenia) or high (-cytosis) blood cell counts, or when cell counts are needed on body fluids such as cerebrospinal fluid (CSF) or seminal fluid

Technical Information

Units of Reporting Cell Counts

The International Committee for Standardization in Hematology [ICSH] recommends the liter (L) as the volume unit to be used when reporting blood cell counts. Thus, cell counts are reported as the number of cells (i.e., wbc, rbc, and plt) per liter of blood. However, you may see counts reported per cubic millimeter (cu mm or mm³) or per microliter (ml). Therefore, you must learn and be prepared to convert data reported in one unit of measure to other units of measure. Remember the following mathematical relationships:

1 mm³ = 1 ml = 10^-6 L Examples of cell count data:

1 x 10⁶ ml = 1 L

WBC 6.5 x 10⁹/L = 6.5 x 10³/ml or 6,500/ml

RBC 4.3 x 10¹²/L = 4.3 x 10⁶/ml or 4,300,000/ml

Methodology

Diluents and Dilutions

Normally, whole blood contains thousands of white cells, hundreds of thousands of platelets, and millions of red cells. In many cases, blood must be diluted before a manual cell count is performed. The diluent used and how large a dilution is made, is determined by the cell(s) to be counted and the method used to perform the count. By convention, physiologic saline (0.85%) is the diluent of choice for a manual RBC. It prevents cell lysis, and inhibits rouleoux. Three per cent [3%] acetic acid is the diluent of choice for a WBC. However, one percent hydrochloric acid or Turk’s diluting fluid may also be used. [Turk’s fluid contains gentian violet which stains the nuclei of wbcs, making them more visible]. These solutions lyse rbcs, making wbcs easier to identify.

One per cent [1%] ammonium oxalate is the preferred diluent for manual platelet counts. It not only lyses rbcs, but it inhibits platelet aggregation and reduces platelet adhesion tendencies. Ammonium oxalate may be used when both a WBC and a PLT are performed simultaneously.

The direct, manual method for counting eosinophils requires the use of phyloxine diluting fluid which contains water, phyloxine, sodium bicarbonate, heparin, and propylene glycol. The phyloxine stains the eosinophils red, while the sodium bicarbonate and water help to lyse all other wbcs. The propylene glycol lyses the rbcs. The addition of heparin prevents cell clumping.

Typically, the dilution for a manual WBC is 1:20, for a manual RBC it is 1:200, for an Eosinophil count it is 1:32, and for a manual PLT it is 1:100. However, in certain conditions, expected cell counts may be extremely high [i.e., leukemoid reaction] or low [i.e., anemia]. In such cases, the dilution made is accordingly adjusted.

Automated cell counters use a variety of brand-specific solutions for making dilutions of blood. For example, Beckman Coulter hematology analyzers use Isotonä, a preserved isotonic solution. Dilutions made are also instrument-specific, but generally are larger than ones made for manual counts [e.g., TOA Sysmexä E-5000 makes a 1:750 dilution for a WBC].

Pipettes [Manual Cell Counts]

Historically, micropipettes are used to make dilutions of whole blood. One such pipette is the Thoma pipette. It comes in two varieties: white cell or red cell. Each pipette is divided into parts or volumes, so indicated by marks on the body of the pipette (figure 1). The stem of each pipette holds a total of 1.0 part while the bulb holds 10 parts [white cell] or 100 parts [red cell]. To make a dilution using these pipettes requires that blood be aspirated first into the stem. Subsequently, as diluent is aspirated, all the blood is pushed into the bulb. To calculate the dilution made only requires that you know to what mark on the pipette stem blood was drawn, and the type of pipette used. For example, if a white cell Thoma is used, and blood is aspirated to the 0.5 mark, then diluted to the 11 mark, a 1:20 dilution is made. [Hint: 0.5 volumes in 10 volumes].

Figure 1. White and Red Cell Thoma pipettes (Steine-Martin et al. 1998)

Today, the Unopetteä is the preferred method for performing timely manual cell counts safely and accurately. The standard Unopetteä system (figure 2) comes with a reservoir containing a set volume of diluent, a self-filling pipette, and a pipette shield. Systems are available for WBC, PLT, RBC, Reticulocyte counts, Eosinophil counts, and for use with automated instruments. Table 1 lists the common Unopetteä diluents, volumes and pipettes used for specific cell counts.

Figure 2. WBC Unopette system with reservoir, pipet, and pipet shield.

1. Protects pipet and is used to puncture the reservoir diaphragm.

2. Is self-filling and size matched with various reservoirs.

3. Contains a pre-measured volume of diluent and is sealed with a plastic diaphragm.

(1) Pipet shield

(2) Pipet

(3) Reservoir

Table 1. Unopette reservoir diluents, volumes, pipet sizes, and final dilutions made.

Test Diluent Pipet , ml Dilution

WBC	3% acetic acid, 1.98 ml 3% acetic acid, 0.475 ml	20 25	1:100 1:20
WBC/PLT	1% Ammonium Oxalate, 1.98 ml	20	1:100
RBC	0.85% Saline, 1.99 ml	10	1:200
Eosinophil	1% Phloxine B, 0.775 ml	25	1:32

Hematocytometers

Automated cell counters employ sophisticated electrical and optical technology to count cells. However, the “sophisticated” technology employed when manually counting cells is called a hemocytometer (figure 3), of which there are three types: Speirs-Levy, Fuchs-Rosenthal, and Neubauer. The Neubauer is the most common type used in for blood cell counts.

Figure 3. A Neubauer hemocytometer. _{(Stiene-Martin et al. 1998)}

Hemocytometers are calibrated glass slides. This means that ruled dimensions are cut into the glass, thus creating a counting chamber (figure 4). In the case of the Neubauer, the dimensions are: (1) length, 3 millimeters; (2) area, 9 square millimeters; and (3) volume, 0.9 cubic millimeters.

Figure 4. Neubauer counting chamber. _{(Stiene-Martin et al. 1998)}

General Principles of Automation

There are three basic principles of operation employed by automated instruments to count cells. They are electronic impedance, light scatter, and centrifugal force. Each principle exploits a physical attribute of cells. For example, electronic impedance technology is based on the fact that cells are poor conductors of electricity, while instruments that measure light scatter depend upon the fact that cells have volume and optical density.

Electronic Impedance

In late 1948, Wallace H. Coulter discovered what has become known as the “Coulter Principle”. This principle takes advantage of the fact that cells are relatively poor conductors of electricity compared to a physiologic electrolyte solution. The early model Coulter counter was composed of: (1) a sensing mechanism made of a small aperture sandwiched between two platinum electrodes immersed in an electrolyte solution; (2) motors, pumps, valves and tubing designed to dilute blood and move suspended cells through the aperture; (3) an electronic signal processing system made of electronic circuits that analyze electrical pulses and counters that count the number of cells within a specified size range. Figure 5 illustrates this “electronic gate” used to count cells.

How does a cell counter count cells accurately? As a cell is pulled through the aperture, a change in the voltage of the sensing system occurs, and a pulse is generated. Each pulse corresponds to a cell and is proportional to the volume of that cell. Cells of different sizes may be discriminated by pulse height analyzers. To eliminate the counting of non-cellular material, the cell counter must be calibrated and thresholds set. By definition, a threshold is a voltage limit with which a pulse is compared. Think of upper and lower thresholds as pulses corresponding to small and large sizes of cells. Manipulating the thresholds permits the establishment of size ranges. Pulses generated by non-cellular material which is above or below the thresholds would be eliminated from the cell count.

Figure 5. The “Heart” of the Early Electronic Gate Cell Counter. _{(Linne’ & Ringsrud, 1999)}

Early model cell counters were referred to as “single-parameter” instruments because they could count only one blood cell type at a time: either red cells, white cells, or platelets. Other cell data was manually calculated. Today, cell counters are “multiparameter”: that is, they are capable of simultaneously counting rbcs, wbcs, plts, and they can carry out complex mathematical calculations to derive hemoglobin, hematocrit, and indices data.

Light Scattering

This technology exploits the fact that cells have optical density. Cells are counted as they pass through a focused beam of light. The light source in this case is a laser (light amplification by stimulated emission of radiation). A typical Optical Gate cell counter is composed of a small-volume flow cell located between a helium-neon laser source and photosensors which are capable of measuring the amount of light scattered by a cell (figure 6).

A cell’s interaction with radiant energy will result in the light waves being bent (diffracted), re-directed back (reflected), and refracted (bent because of a change in speed). Relative to cell counting by light scatter, it has been observed that light is scattered in all directions when intercepted by a cell, that diffraction is the predominant event in the acute angles relative to the incident light, that reflection occurs predominantly at the obtuse angles, and refraction generally occurs at intermediate angles. This knowledge is also used to differentiate white cells types: a lymphocyte from a neutrophil from a monocyte.

Figure 6. Optical Gate Cell Counter. _{(Stiene-Martin et al. 1998)}

Centrifugal Analysis

When whole blood is centrifuged, constituents separate forming layers (figure 8) based on the specific gravity of each component. Becton Dickinson’s QBCâ [quantitative buffy coat] instrument (figure 9) uses an optical device to measure the lengths of the layers (figure 10). This method of centrifugal analysis was first described in the 1980’s, and the instrument is designed primarily to be used in physician office laboratories (POL).

Figure 8. Centrifugal effect on whole blood. Figure 9.

Figure 10. QBC Blood Tubes and Analysis

Monocytes & Lymphocytes

top

bottom

Red cells PMNs Platelets Cell-free plasma

_{(Becton Dickinson and Company , 1996)}

How does it work?

A specially manufactured capillary tube is pre-coated with the supravital stain acridine orange. Non-granulocytic white cell nucleoprotein absorb the stain and fluoresces green when exposed to violet light. Granulocytic cells contain many glycosaminoglycan-containing granules; the stain absorbed by these granules fluoresces at a different wavelength than nucleoproteins. The combined nuclear and granule fluorescence causes the granulocytic layer to appear bright orange in color. Platelets appear yellow and form the top cell layer. Red cells contain iron. As the heaviest cells, they form the bottom layers within the capillary tube following centrifugation.

QUALITY ASSURANCE IN THE HEMATOLOGY LABORATORY

Every measurement made in the hematology laboratory contains inherent variability. This variability results from error: random and systematic. While random error affects precision or test reproducibility, systematic error always affects accuracy or how well a measured value agrees with the true value. Since physicians and other healthcare providers depend on the hematology laboratory for tests results that are used in making critical decisions regarding their patients, the hematology laboratory must take what steps are necessary to control error and ensure the quality of the analyses.

A quality assurance program [QAP] is the way any laboratory can guarantee the quality of its analyses. A quality assurance program is designed to monitor and evaluate the total testing process [preanalytic, analytic, and postanalytic], the identification and corrective measures for problems, the testing procedures and results reporting, and the adequacy and competency of the people performing the tests. Elements of a quality assurance program include written policies and guidelines, technical procedure manuals, quality control [QC], proficiency testing [PT], equipment maintenance programs, patient test management and record-keeping, continuing education and safety training for lab personnel, and inspection and accreditation of the laboratory.

The FGCU CLS hematology student laboratory has a quality assurance program. It is different from conventional because testing is done for training purposes, not diagnostic. It is outlined in table 3 below.

Table 3. Comparison of the FGCU CLS Student Hematology Lab with a Standard Hospital Hematology Lab

Lab Type	Written Policies & Guidelines	Technical Procedure Manuals	Quality Control	Proficiency Testing	Equipment Maintenance Program	Test Manage-ment	Training & Safety for Lab Personnel	Inspection & Accreditation
Hospital Hematology Lab	Mission Statement, Safety Manuals, PM Records	Procedure Manuals	Calibrators, Controls, Establish and use limits	CAP, AACC, Others	Biomedical Department, daily, weekly, monthly, semi-annual & annual PM	LIS, Mainframe	Ongoing	CAP, HCFA, State
FGCU CLS Student Hematology Lab	Course Web Page, Syllabus, Course Texts, Safety Manual	Course Syllabus, Course Textbook	Calibrator, Controls, Use established limits	Lab Exercises, Tests, Quizzes, Lecture discussions	Course Instructor, Students, Day of Use	Written Lab Reports	Course	University, State, NAACLS

It is expected that each student will actively participate in the student hematology laboratory QA program by attending class, asking questions, engaging in class activities, and by using available resources.

Exercise 1: Automated Cell Count

Directions.

1. Using the Coulter Maxmä, perform a red cell count on the specimen provided.

2. Attach the results printout to your data sheet.

3. Reference procedure found at the instrument station.

4. QA & QC: see procedure found at instrument station.

Exercise 2: Manual cell Count

Directions.

1. Perform a manual red cell count on the specimen provided.

2. Record data on the report form provided.

3. Reference Unopetteä procedure.

4. QA: (1) Check sample for clots; (2) Use clean, intact hemocytometer; (3) Check volume level of diluent in reservoir; (3) Check pipette for cleanliness and absence of chips or cracks; (4) Select correct size pipette for type of diluent used and cell count to be performed; (5) Use scrupulous technique, including the “battlement” counting pathway.

5. QC: Counts are performed in duplicate [i.e., set up two unopettes for each count]. There should be no more than a 10-cell difference between the highest and lowest total number of cells counted within the 5 secondary squares counted. Total cell counts on each side of the counting chamber should agree within 10% of each other. Accuracy = +20% of automated for RBC.

REFERENCES:

1. Campbell M, Biochemistry, 2nd ed., Saunders College Publishing, 1995. http://www.stark.kent.edu/~cearley/pchem/Review1/globpic.htm

2. Harmening D. Clinical Hematology and the Fundamentals of Hemostasis, 3^rd ed., Philadelphia, FA Davis, 1997.

3. Linne’ JJ and Ringsrud KM, Clinical Laboratory Science: The Basics and Routine Procedures, 4^th ed., St. Louis, Mosby, 1999.

4. Stiene-Martin EA, Lotspeich-Steininger CA, and Koepke JA: Clinical Hematology Principles, Procedures and Correlations, 2^nd ed. Philadelphia, Lippincott, 1998.

Red Cell Count Data Report Form

Student Name: ________________________ Date: _______________

Specimen ID: _____________ Date & Time Collected: _______________

Data

Exercise 1. Staple data printout to this form.

Exercise 2. Record data in blanks below.

Manual Counts QAP Data

Unopetteä Lot # __________

Pipette size used __________

Diluent used __________

Formula: Number of cells counted x dilution factor _{= cell count/ml}

Number of squares counted x area x volume

Unopette #1		Unopette #2
Criterion	Side 1	Criterion		Side 2
Total # cells counted		Total # cells counted
Counts agree + 10% {yes or no}
Highest # cells counted		Highest # cells counted
Lowest # cells counted		Lowest # cells counted
Highest – Lowest #		Highest – Lowest #
Difference {Std = +10 cells }		Difference {Std = +10 cells }
Area		Area
Volume		Volume
Dilution factor		Dilution factor
Cell Count		Cell Count
Count Average {cells/ml} ^Manual
Red Cell Count ^Automated
Accuracy {Std = +20% Automated}
Reference Range	Adult Male 4.6 – 6.2 x 10^12/L Adult Female 4.2 – 5.4 x 10^12/L
QC Checks [Failure to meet standard requires that the procedure be repeated to correct for errors].			Clinical Significance [Data above or below ranges may* indicate illness or disease]. *Assuming QC within limits

STUDY QUESTIONS

1. List four sources of error associated with the use of a hemocytometer.

2. Describe two situations that require the repeating of a manual cell count.

3. You inadvertently counted the red cells in five primary squares of a Neubauer hemacytometer. The sample dilution used was 1:200. The number of cells counted: 345. What is the red cell count in cells/mL? cells/ml? cells/L