How to do a manual WBC differential count, and the battlement method

What the count is, and when it is done

A differential white cell count reports what proportion of the white cells present belong to each class — neutrophils, lymphocytes, monocytes, eosinophils, basophils, and any immature or abnormal forms seen. Modern analysers produce a differential automatically on most samples. The manual count exists for the cases where the analyser flags something, cannot classify what it is seeing, or where morphology itself is the question.

That is worth stating plainly, because it frames everything below: the manual differential is the method used precisely when the answer matters and the machine would not do.

Two kinds of cell on the film fall outside the five classes. Immature and abnormal white cells, such as blasts, are white cells, so they belong in the count under a class of their own. Nucleated red cells are not, so they are tallied beside the count and reported per 100 white cells, which is the figure used to correct an analyser’s white count for them. The differential counter has a key for each, and does that correction from the count you type.

The hardest call in the count is the band: band vs segmented neutrophil sets out the filament rule, the one-third rule and how often observers disagree. Once the differential is counted, the absolute neutrophil count is the white count times the segmented neutrophils and bands added together.

Where on the film to count

Not anywhere. A blood film is not uniform, and where you count changes what you count.

The count is made in the monolayer — the region where red cells lie in a single layer, touching but not overlapping. Too near the head of the film the cells are piled up and unreadable. Too near the tail and the distribution is distorted: larger cells, particularly monocytes and neutrophils, are dragged toward the edges and the feathered end, while lymphocytes are relatively over-represented in the middle. Counting in the wrong region does not add random noise; it adds a bias in a predictable direction, which is worse.

The monolayer only exists on a well-made wedge smear, and the same monolayer is where a platelet estimate is counted.

The battlement method, and why

The battlement method is a fixed path for choosing which fields of a blood film to count in a differential: a few fields along the edge of the film, a few inward at right angles, a few parallel to the edge again, then back out to the edge, so the track looks like the top of a castle wall. Every field on the path is counted completely before moving on.

The reason is the bias above. A straight track along the film samples one band of it and inherits whatever distortion that band carries. The battlement deliberately crosses between the edge and the centre repeatedly, so that over-represented and under-represented regions are sampled in turn and their biases partly cancel. The pattern is not ritual, it is a sampling correction.

Where it comes from: the 1940 comparison

The method was set against its rivals by R. G. Scott MacGregor, W. Richards and G. L. Loh in “The differential leucocyte count”, The Journal of Pathology and Bacteriology 51(3), 1940, pages 337–368 (doi:10.1002/path.1700510303). They compared the three tracks most used at the time — the straight edge count, the cross-sectional count and the battlement — and found the battlement gave the most accurate results. Their definition, as quoted in a University of Pretoria dissertation on the differential leukocyte count, is a “count made of three horizontal edge fields followed by two fields towards the center (so as to give three vertical fields), followed by two fields in a horizontal and then two fields in the vertical direction again”.

The same dissertation records that the battlement is the method recommended by the National Committee for Clinical Laboratory Standards (now CLSI), and that its reference differential examines each film “using the ‘battlement’ method”, with two hundred white cells counted on each film.

“Battlement” is not one path

Ask how far in the track goes and the sources disagree. The 1940 version turns two fields in from the edge; teaching notes describe an exaggerated battlement that goes a third of the way across the film, and meander methods that split the film into two or four parts and repeat that path in each. The search results for “battlement method” mix these freely, which is why they read as contradictory. They are different tracks with the same purpose.

The lecture-note descriptions are from Lecture Notes in Medical Technology, lecture 13. Where a laboratory’s procedure names one track, that is the one to use; the comparison above is for reading the literature, not for overriding a standard operating procedure.

The mechanics the descriptions leave out

Three things decide whether a battlement count is actually a battlement count, and none of them is the shape. Move one whole field at a time, so the next field starts where the last one ended: overlapping fields count cells twice, and gaps skip them. Count every white cell in a field before moving, including the awkward one at the rim, applying the same rule each time for cells cut by the edge of the field. And stay inside the monolayer: when the inward leg would run into the thick part of the film, turn early rather than counting where the cells overlap.

The track also has an end condition the shape does not supply. On a film with a low white count a single pass may not reach a hundred cells; continue the same pattern further along the film rather than doubling back over fields already counted.

How many cells: 50, 100, 200

One hundred cells is the routine count, and it is chosen because the arithmetic is trivial: with a denominator of 100, the count of each class is already its percentage. The number is a convenience as much as a standard.

The classical guidance is to begin with a hundred and extend to two hundred when circumstances require it — a rare population to pin down, or a raised white count where a small shift matters. The reference standard of two examiners counting two hundred cells each is rarely run outside method comparison work, for obvious reasons of workload.

The imprecision built into the method

This is the part that is easy to work with for years without internalising, and it is not a matter of care or skill. Classifying a sample of cells is a sampling problem, and sampling has irreducible error.

The counts follow a Poisson distribution, and the coefficient of variation is approximately 1 ÷ √n. At a hundred cells that is 10%, before anyone makes a single mistake. Not 10 percentage points — 10% of the figure — but the effect on an individual class is easier to see written out.

Read the hundred-cell row. A monocyte figure reported as 6% is consistent with anything from about 1% to about 11%, purely from having counted a hundred cells rather than all of them. That is the method behaving correctly. The same square-root rule sets how many cells to count on a hemocytometer, and, as in a McMaster faecal egg count, precision follows the number actually counted.

Two consequences follow, and both are practical:

• Small differences between two differentials are usually not differences. A move from 6% to 9% on successive hundred-cell counts is comfortably inside what sampling alone produces.
• Doubling the count does not halve the error. Because the error goes with √n, going from 100 to 400 cells — four times the work — halves it. That is the real reason the reference method is expensive and routine practice is not.

None of this is an argument against the manual differential. It is an argument for knowing what a percentage from it does and does not settle.

Keeping the tally without losing the field

The mechanical problem is small but real: your eyes are at the eyepieces and your hand is on the stage, so anything that requires looking away costs you your place in the field.

That is what the traditional multi-key laboratory tally counter solved — one key per class, found by touch, with a bell at a hundred. The same shape works on a keyboard: a key per class, a count that stops on its own at the target, and percentages calculated for you so no arithmetic happens at the end. What matters is that recording a cell takes no attention, because the attention belongs at the eyepiece. A reticulocyte count keeps two tallies the same way: the Miller disc method.

What no counter does is the identification. Classifying the cell is the skilled part and the whole point; the counter holds the tally and nothing more. It carries no reference ranges, makes no comment on whether a result is expected, and is not a diagnostic aid. Reference intervals are the property of the reporting laboratory (published adult ranges are compared in WBC differential normal ranges), and interpretation belongs to the clinician and the laboratory professionals responsible for the sample.