TITLE: Differential Leukocyte Count

PRINCIPLE: The purpose of a microscopic differential count is to establish the relative frequency of each type of cell. The microscopic differential leukocyte count on peripheral blood follows a definite route, starting at the thin feathered edge, where RBC’s just start to overlap, and continuing into the thicker edge of the smear, moving over one field at a time.

A differential of the white blood cells, a review of the RBC and platelet morphology, in a blood sample provides the physician with information on a patient’s condition. It is important that technicians and technologists “read” differentials the same way within the department.

Periodically we have slides of interest (malignant cells, immature cells, abnormal red cell morphology) for review by all Techs.

Annually, staff reviews a minimum of one set of 10 CAP photographs as a means of competency. The CAP photographs are graded according to the CAP Participant Summary Report. Staff not meeting the 80% or above the CAP standard receive a Survey Exception Form. Staff must redo/review and return the form along with their revised photograph identifications to the Senior Tech. The Senior Tech re-grades the form and follows up with appropriate remedial competency as needed. Performance is kept in their file for annual competency. When staff performance results in a Survey Exception Form, the staff member is required to participate in the next set of 10 CAP photographs.

Annually, one slide is selected for all staff to perform a manual differential and RBC morphology. Staff performance is compared for the manual WBC differential and RBC morphology. WBC parameters should agree within 10% of the Senior Tech performed differential. RBC morphology should agree within 1+ of the Senior Tech performed differential.

A chart of morphology is at each scope so that consistent grading of morphologic features is reported by all personnel. We have a double-headed scope for review of unusual or difficult cases.

PERSONNEL

Medical Technologists and Technicians

SPECIMEN TYPE:

Specimens collected in K2 EDTA.

REAGENTS AND EQUIPMENT:

Wright Giemsa Stained (Peripheral smears)

QUALITY CONTROL:

Daily stain QC is performed by staining the first slide of the day with a WBC > 2.0.

The stain on this slide day is compared to the stain quality on the QC slide (slides made, stained and cover slipped after monthly maintenance).

Daily slide quality is documented on the Aerospray Preventative maintenance chart (Attachment A).	A. If the slide stain qualities are comparable, the Daily Control slide box is checked on the Preventative maintenance chart (Attachment A) no further action is required.

B. If the slide stain qualities are not comparable:

1. Check reagent levels and replace when necessary.

2. Perform a carousel clean or Daily Maintenance if it has not been performed for your shift.

3. Notify Senior Tech if the previous steps have not resolved the stain quality.

Document on the Aerospray Preventative maintenance chart under “Corrective Action Log”

STEPWISE PROCEDURE:

1. A.	On 50x oil, count 100 WBC’s. Refer to a glossary of cellular identification if necessary. Count the cells in an area of the slide where the red cells are relatively close together but not overlapping.

Note WBC morphology: any vacuoles, toxic granulation, Dohle bodies or hypersegmentation present in the WBC’s is included with the reported differential.

Report the NRBC’s: If there are NRBC’s, report that number as a part of the manual differential. We report the “Corrected WBC Count” from the analyzers which corrects for NRBC’s and Cellular Interference. If the WBC estimate does not match the Corrected WBC count from the analyzer, manually calculate the Corrected WBC using the NRBC’s present in the differential and the Uncorrected WBC count from the analyzer. If that calculation matches your estimate, enter that result in the WBCI field and save. WBC x 100/NRBC + 100 = Corrected WBC count.

Do a WBC estimate: If there are 5 or more Giant Platelets, micro megakaryocytes or megakaryocytes present in the differential count to verify the WBC Count. A manual count should be done if the WBC estimate and the corrected WBC Count do not match,

perform a manual WBC/PLT count and compare with your WBC/PLT estimate on the smear. Report your manual count (WBCI and PLT). Document on Manual Count Log.

2. A.	Under 100x, do a platelet estimate. Refer to procedure 4840-HE-0335, “Platelet

Estimate from a Stained Blood Smear”. Along with the platelet estimate, a canned message stating that the platelet morphology is normal or abnormal is attached to the estimate result field. Any abnormal platelet morphology is to be reported as part of the differential. Add the type of abnormal morphology using the comment function and the canned messages we have built in the system for most cases, or free text can be used in the comment box.

Abnormal platelet morphology includes, but is not necessarily limited to: giant platelets, hypogranular platelets, platelet satellitism, EDTA platelet clumping phenomenon or an increased variability in platelet size known as platelet anisocytosis.

Under 100x, examine RBC’s for morphology, look for:

Anisocytosis: The degree of variation of cell size from one to another. A small amount of variation is normal.

Grade as 2+ to 4+. (See following page). The RDW is an indicator of anisocytosis. Roughly, an RDW (red cell distribution width) of 14.6-16.4 is about 1+ anisocytocytosis, 16.5-19.0 about 2+, 19.1-21.9 about 3+, greater than 22.0 about 4+. The Coulter sees a 3- dimensional picture of red blood cells in isotonic saline. Under the microscope we see a 2-dimensional picture of dried red blood cells. Artifacts do occur. Remake slides if the RDW and the manual slide are very different from each other.

Cell Size: The MCV (Mean Cell Volume) is used to aid in identifying cell size. If the MCV is greater than normal, macrocytes are probably present. If the MCV is less than normal, microcytes are probably present, a significant number of microcytes or red cell fragments requires a platelet estimate from the smear to verify the numerical value from

the analyzer. Occasionally the MCV may be normal but both macrocytes and microcytes may be present.

Grade as 2+ to 4+ (see following page). A MCV of 75-79 is 1+ micro, 70-74 about 2+, 64-69 about 3+ and less than 63 about 4+. An MCV of 96-101 is 1+ macro, 102-110 about 2+, 111-120 about 3+, and greater than 121 about 4+.

Quantitate the type of misshapen cells: teardrop cells, sickle cells, etc.

Relative Hemoglobin Content: Hemoglobin content of the cell is

reflected by the central pallor; the normal red cell has a central pallor that is one-third of the entire cell.

Evaluate hypochromia by the number of cells that are hypochromic and

how severe it is in each cell. The MCH is also an indicator of hypochromasia. (See following page).

Polychromatophilia: Immature RBC’s stain “bluer” than mature RBC’s.

Grade according to accompanying table. (1+ to 4+).

Inclusions:

Pappenheimer, Howell-Jolly bodies, parasites are all noted as being “present”. They need not be quantitated unless many are present per field.

Basophilic stippling is graded by the numbers of cells per field. (This does not include that seen in polychromatophilic RBC’s). (See following page).

Rouleaux or Agglutination:

Rouleaux appears as stacked coins. Rouleaux often runs up and down the slide. Agglutination tends to go left and right across the slide. Beware of confusing agglutination with rouleaux. Prepare another slide before reporting rouleaux. State agglutination or rouleaux as “present”.

Double cell populations should be recognized and reported as such:

The RDW and the RBC histogram of the Coulter will identify double cell population.

If there are no RBC abnormalities, there is mnemonic in LIS morphology- “RBCM” – use this and enter “Normal”. The patient report will state RBC morphology is normal.

DIFFERENTIAL LEUKOCYTE COUNT:

RBC MORPHOLOGY

1+ Macro	MCV 96-101	1+ Baso. Stippling	1-3 Cells
2+ Macro	MCV 102-110	2+ Baso. Stippling	4-6 Cells
3+ Macro	MCV 111-120	3+ Baso. Stippling	7-10 Cells
4+ Macro	MCV > 120	4+ Baso. Stippling	>10 Cells
1+ Micro	MCV 75-79	1+ Poly	1-3 Cells
2+ Micro	MCV 70-74	2+ Poly	4-6 Cells
3+ Micro	MCV 64-69	3+ Poly	7-10 Cells
4+ Micro	MCV < 64.0	4+ Poly	> 10 Cells
1+ Hypo	MCH 25-27	1+ Schistos, acanthos, burrs, sickles, spheros	1-3 Cells
2+ Hypo	MCH 21-24	2+ “	4-6 Cells
3+ Hypo	MCH 21-24 MCHC 29-30	3+ “	7-10 Cells
4+ Hypo	MCH <21 MCHC 25-28	4+ “	> 10 Cells
1+ Aniso	RDW 14.6-16.4	1+ Elliptos, targets, tears, stomatocytes	2-10 Cells
2+ Aniso	RDW 16.5-19.0	2+ “	11-20 Cells
3+ Aniso	RDW 19.1-21.9	3+ “	21-30 Cells
4+ Aniso	RDW > 22.0	4+ “	> 30 Cells

4.	Slides are retained for 4 weeks in the slide storage box. Significant abnormal results are

to be reviewed by the senior tech or pathologist. See procedure 4840-HE-0100 “Review of Abnormal Hematologic Findings”.

CALCULATIONS

None

REPORTING AND INTERPRETING RESULTS:

Report results through the Laboratory Information System. Refer to the LIS Procedure Manual for complete instructions.

NORMALS

Normal reference ranges print on the patient report, and are listed in the LIS, they are age related. The instrumentation has normal ranges for age groups built in the workstation.

PROCEDURAL NOTES

The technologist is required to request that the Senior Technologist in Hematology or in lieu of the Senior Technologist one of the Pathologists review all peripheral blood smears which in her/his opinion is uncertain of the cellular identification or interpretation.

REFERENCE

Miale, John B., M.D., Laboratory Medicine Hematology , 3rd

Ed. Pg. 1130, 1967.