# Hematology Laboratory

A*t the end of this chapter, the reader should be able to do the following:*

1. Estimate the hematocrit if given the hemoglobin using the rule of three and vice versa.

2. Estimate the hemoglobin if given the red blood cell count.

3. Calculate red blood cell indices (MCV, MCH, MCHC).

4. Correct the WBC count for the presence of nucleated red blood cells.

5. Calculate white blood cell, red blood cell, and platelet counts performed on a hemacytometer for blood and body fluids.

6. Calculate the sperm concentration per milliliter.

7. Calculate the sperm count per ejaculation.

8. Calculate the number of reticulocytes by both the slide and Miller disk method.

9. Correct the reticulocyte count for anemia using the reticulocyte index.

10. Correct the reticulocyte count when increased reticulocyte production is present.

## RULE OF THREE

Hematology is the study of the cells within the blood. These include the white blood cells (WBCs), platelets, and the cells in the largest quantity, the red blood cells (RBCs). The RBCs carry oxygen from the lungs to the tissues and carbon dioxide from the tissues back to the lungs where it is exhaled. The oxygen is carried by hemoglobin molecules. The quantity of hemoglobin can be determined spectrophotometrically by automated hematology instruments. If the hemoglobin concentration is low, the oxygen-carrying capacity of the RBC is decreased as well. This will lead to the condition called anemia. The most common cause of anemia is iron deficiency. Iron is vital to the structure of the hemoglobin molecule, and if the iron concentration is low, the hemoglobin concentration will be adversely affected.

A second very common hematology test that is performed is the hematocrit. This is the ratio of the blood cells to plasma expressed as a percent. The reference range for hematocrit is 37% to 47% for adult women and 42% to 52% for adult men, meaning that approximately half of the whole blood in the body is made up of cellular components; the remainder is the liquid plasma portion. If the number of RBCs is decreased, the hematocrit will be decreased. If a person is anemic, the hemoglobin and hematocrit will both be decreased. The adult reference range for hemoglobin is 12-18 g/dL for men and 12-16 g/dL for women.

There is a relationship between the hematocrit and the hemoglobin values that is useful as a quality assurance tool. The hematocrit is usually three times the hemoglobin value plus or minus 3. This is known as *the rule of three*. If the hematocrit is known then the hemoglobin value can be estimated and vice versa. This is a good rule of thumb to keep in mind even when the hemoglobin or hematocrit is either below or above the normal range. When the rule of three is not found, it may indicate an error or an abnormality in the size of the RBC or the amount of hemoglobin in the RBC. This error may be an instrument malfunction of some sort, a mix up in patient identification, a problem in reporting the correct result, or a problem in the patient’s RBCs.

###### Example 9–1

A finger stick was performed on a young boy in a pediatrician’s office. The medical laboratory technician performed a spun hematocrit while the medical assistant performed the hemoglobin using a CLIA-waived hemoglobin instrument. The hematocrit result was 45% and the hemoglobin was 11 g/dL. Are these results consistent with the rule of three?

No, these results do not follow the rule of three. The hemoglobin should be 15 g/dL if the hematocrit is accurate, or the hematocrit should be 33% if the hemoglobin was accurate. The medical laboratory technician should investigate the discrepancy.

The hemoglobin would be estimated to be **12** g/dL.

The hematocrit would be estimated to be **15** g/dL.

The rule of three can also be used to estimate the hemoglobin values using the RBC count. The RBC count × 3 should equal the hemoglobin value. The adult reference range for RBC count is 4.7-6.1 (10^{12}/L) for men and 4.2-5.4 (10^{12}/L) for women. This additional relationship can also be used as part of the laboratory’s quality assurance plan because errors may indicate pre-analytical or analytical errors or a condition such as anemia in the patient.

## RED BLOOD CELL INDICES

The size of the RBC is important in the treatment of anemias. Some anemias cause the RBC to be smaller than normal; others cause it to be larger. The relationship among size, hemoglobin, and hematocrit was mathematically determined by Wintrobe in the 1920s. Three formulas were developed to describe RBC morphology and to aid in the classification of anemia.

### Mean Corpuscular Volume

The first formula is the mean corpuscular volume (MCV). The MCV describes the average volume or size of the RBC in femtoliters (fL) and is calculated by the following formula:

The normal range for MCV is 80 to 100 fL. MCV values less than 80 fL suggest RBCs that are smaller in size, whereas MCV values greater than 100 fL suggest RBCs that are larger than normal.

###### Example 9–3

Calculate the MCV of a sample if the hematocrit is 45% and the RBC count is 4.5 (10^{12}/L).

Use the formula to calculate MCV:

The MCV of this sample is **100** fL.

### Mean Corpuscular Hemoglobin

The second formula is the mean corpuscular hemoglobin (MCH). As the name implies, it represents the average amount of hemoglobin present in the individual RBC in units of picograms (pg), significant only to the nearest tenth. It is calculated by the following formula:

The normal range for MCH is 27 to 31 pg.

### Mean Corpuscular Hemoglobin Concentration

The third calculation is the mean corpuscular hemoglobin concentration (MCHC). This calculation is a ratio of the hemoglobin to the hematocrit and is expressed in terms of percent and significant only to the nearest tenth. The MCHC represents the average hemoglobin content in the patient’s RBC population.

The normal range for MCHC is 32% to 36%.

### Red Cell Distribution Width

Red cell distribution width (RDW) is a measurement of the degree of anisocytosis present, or the degree of variability in RBC size, in a blood specimen.

Reference range is 11.5% to 14.5%.

This calculation is usually performed by the automated hematology analyzers but is presented here as it is very commonly used in the hematology laboratory.

## CORRECTION OF THE WBC COUNT FOR NUCLEATED RBCs

Nucleated RBCs (NRBCs) are immature RBCs that have not lost their nucleus. They are also called *orthochromic normoblasts* or *metarubricytes*. They are the last immature RBC that contains a nucleus, and when they further mature they become reticulocytes. Normally the NRBC is found only in the bone marrow. In times of severe anemic stress they may be found in the peripheral blood and they are also normally found in the peripheral blood of healthy newborns but disappear after a few days. Premature infants will have NRBCs present at birth and they may be present longer than a week.

Since NRBCs contain a nucleus, the WBC automated cell count should be adjusted downwards as the instrument may count the NRBC nucleus as a WBC. A correction calculation is usually performed when greater than 5 NRBCs per 100 WBCs are seen on a peripheral smear.

The formula to correct for NRBCs is:

###### Example 9–6

A CBC with manual differential was ordered for a premature newborn born at 36 weeks gestation. Twenty-six NRBCs were counted in the differential. The baby’s WBC count was 27.5 (10^{9}/L). What is the corrected WBC count?

To solve this problem, use the above formula:

## CELL COUNTING BY THE HEMACYTOMETER METHOD

The majority of RBC, WBC, and platelet counts are performed by automated hematology analyzers. Occasionally, the count may be too low for the instrument to count the cells accurately. WBCs in body fluids are often counted manually. In these instances, cells are counted with the aid of a microscope using a counting chamber called a hemacytometer (Figure 9–1). The most common hemacytometer used in clinical hematology is the Neubauer hemacytometer. This hemacytometer consists of two identical counting chambers. Each chamber contains an etched grid constructed to have a total surface area of 9 mm^{2}. The chambers are identical, so that a sample can be counted in duplicate using the same hemacytometer. The count from each chamber should be within 10% of each other to ensure an accurate count. A dilution of whole blood is used and a small amount of diluted sample is allowed to flow between the coverslip and hemacytometer chamber. The distance between the coverslip and the surface of the hemacytometer is 0.1 mm.

Figure 9–1 is a schematic of a Neubauer hemacytometer grid in which each of the large squares is numbered 1 to 9. WBCs are counted in the four corner squares (squares Nos. 1, 3, 7, and 9), whereas platelets and RBCs are counted in the central square (square No. 5). Each large square is 1-mm^{2} in area. The large squares are further divided into 16 smaller squares to facilitate ease of counting the WBCs. The center square is divided into 25 small squares. Each of the 25 small squares is further divided into 16 smaller squares for RBC and platelet counting. The small square A is shown enlarged in Figure 9–2.

### White Blood Cell Count

When the WBC count is abnormally elevated or decreased or the quantity of WBCs in fluid is requested, the hemacytometer method may be used to quantify the total WBC count. Using a micropipette, a ^{2}, and the depth is 0.1 mm. Therefore, the volume in each large square is 0.1 mm^{3} (volume = area × depth).

The mathematical formula to calculate the number of cells per cubic millimeter is as follows:

Area counted = number of large squares counted

Depth factor = reciprocal of depth [1/(1/10)] = 10

Dilution factor = reciprocal of dilution

The WBC count reference range in adults is 4–11 (10^{9}/L)

###### Example 9–7

A WBC count was performed on a sample that was abnormally decreased below the linearity of the automated cell counter. A

In this problem, all eight squares were counted; therefore, the area counted would be 8. The dilution factor would be 10, as a

For many WBC counts, the dilution performed is ^{3} of blood ^{3} (^{3}

The mathematical formula to calculate the factor is as follows:

Factor = 1/area × depth factor × dilution factor

Area counted = number of large squares counted

Depth factor = reciprocal of depth [1/(1/10)] = 10

Dilution factor = reciprocal of dilution = [1/(1/20)] = 20

###### Example 9–8

A WBC count was performed using a hemacytometer and a

Multiply the amount of cells counted by the factor of 50:

Therefore, the WBC count is **7000**/mm^{3}. There are three ways that WBCs might be reported. As mm^{3} is equal to 1 μL, the count may be reported as 7000/μL. In addition, 1 fL is equal to 1000 μL, so many laboratories may report the count as 7.0 × 10^{9}/L.

A manual WBC count was performed by a medical laboratory technician/clinical laboratory technician student in a student laboratory experiment. The four large squares of one side of a hemacytometer were used, and the dilution used was

Since the four large squares were used, and the dilution was

A manual WBC count was performed by a medical laboratory technologist/clinical laboratory scientist student in a student laboratory experiment. The four large squares of one side of a hemacytometer were used, and the dilution was

Since the four large squares were used, and the dilution was

### Red Blood Cell Count

As there are so many more RBCs than WBCs, the cells are diluted ^{2} (0.2 mm × 0.2 mm). Because five of these squares are counted, the total area counted is 0.04 × 5, or 0.2 mm^{2}. Manual RBC counts in EDTA-anticoagulated blood are rarely performed in the United States but may still be used in educational settings as a method to teach the use of the hemacytometer, and in laboratories outside of the United States. Manual RBC counts in fluids such as spinal fluid are performed and are covered in the fluid section of this chapter. As in WBC counting, a factor can be calculated and used to easily quantify RBCs.

The formula to calculate the factor for RBC counts is as follows:

Factor = 1/area × depth factor × dilution factor

Area counted = area of small squares counted = 0.2 mm^{2}

Depth factor = reciprocal of depth [1/(1/10)] = 10

Dilution factor = reciprocal of dilution = [1/(1/200)] = 200

###### Example 9–9

A RBC count was performed on a sample that was diluted

Five hundred and fifty-five RBCs were counted in the 80 small squares used for RBC counts. What is the total RBC count in terms of 10^{6}/mm^{3}?

Using the factor method to solve this problem, the factor is equal to 10,000. As 550 cells were counted, then the RBC count =10,000 × **550** = 5,500,000 or **5.5 × 10 ^{6}**/mm

^{3}

### Platelet Count

Platelets are small fragments of cells found in the bone marrow called *megakaryocytes*. Platelets function in the initial phase of the coagulation process. The reference range for platelets is 150–400 (10^{9}/L). All 25 squares in the central square of the hemacytometer are used to count platelets. Remember, that within each of the 25 squares, there are 16 smaller squares, so the total amount of squares counted for platelets will be 400. As each of the 25 squares has an area of 0.04 mm^{2}, and all 25 squares are counted, the total area counted is 1 mm^{2} (0.04 × 25). A

The mathematical formula for the factor for platelets is as follows:

Factor = 1/area × depth factor × dilution factor

Area counted = area of small squares counted

Depth factor = reciprocal of depth [1/(1/10)] = 10

Dilution factor = reciprocal of dilution = [1/(1/100)] = 100

###### Example 9–10

A platelet count was performed using a

The factor for platelets is 1000. As 150 platelets were counted, the total platelet count is as follows:

A platelet count was performed using a

The factor for platelets is 1000. Therefore, the platelet count is 1000 × 85 or **85,000**/mm^{3}.

A platelet count was performed using a

## BODY FLUIDS

RBC, WBC, and platelet counts are not the only cell counts performed in the hematology laboratory. Cells from CSF, transudates, and exudates, along with semen analysis may be performed. The hemacytometer is used to count both RBCs and WBCs in the different types of fluids and to provide both qualitative and quantitative sperm counts.

### Cell Counts for CSF Specimens

When a spinal tap is performed, three sterile tubes are collected and labeled 1, 2, and 3 in the order they are drawn. The first tube (Tube 1) will be sent to the chemistry laboratory for glucose and total protein analysis, and/or serology laboratory; Tube 2 will be sent to the microbiology laboratory for culture; and Tube 3 will be sent to the hematology laboratory for WBC and RBC counts. CSF cell counts should be performed within 1 hour of collection for the most accurate results. If that is not possible, the CSF tubes for hematology should be refrigerated. Some hematology analyzers, but not all, can perform cell counts on CSF and other body fluid specimens, which eliminates the need for manual cell counts.

### Cell Counts for Synovial, Pleural, Pericardial, and Peritoneal Fluid

Samples for these fluids are usually collected in a heparinized or an EDTA tube. Table 9–1 contains the body fluid normal results.

TABLE 9–1

CSF | Serous (Pleural, Pericardial, Peritoneal) | Synovial | ||

Adult | Neonate | |||

Appearance | Clear and colorless | Clear and colorless | Pale yellow and clear | Pale yellow and clear |

RBC |
0 to 1/mm^{3} |
0 to 3/mm^{3} |
0 to 1/mm^{3} |
0 to 1/mm^{3} |

WBC |
0 to 5/mm^{3} |
0 to 30/mm^{3} |
0 to 200/mm^{3} |
0 to 200/mm^{3} |

Neutrophils (includes bands) | 2% to 6% | 0% to 8% | <25% | <25% |

Lymphs | 40% to 80% | 5% to 35% | <25% | <25% |

Monocytes | 5% to 45% | 50% to 90% | Included with others | Included with others |

Others | Rare | Rare | Monocytes and macrophages 65% to 75% | Monocytes and macrophages 65% to 75% |