MICRO HEMATOCRIT
PRINCIPLE
The micro hematocrit represents the volume of circulating blood that is occupied by erythrocytes and is expressed as a percentage. The micro hematocrit test helps the medical provider identify patients at risk of developing anemia and manage patients who have anemia. Anemia is a less than normal number of erythrocytes in the volume of packed cells. The most common cause of anemia throughout the world is iron deficiency. A decrease in the micro hematocrit value results in a lack of energy, a reduced resistance to infections, and an increased risk of abnormality in pregnancy. An increased micro hematocrit value can result from polycythemia, hemoconcentration, and/or cardiac disease.
SUPPLIES AND EQUIPMENT
1. Equipment
High-speed centrifuge
Micro hematocrit tube reader
Timer
2. Materials/Reagents
Capillary tubes (heparinized) if finger stick specimens used
Capillary tubes (nonheparinized) if whole blood is used
Clay to seal tubes
Laboratory tissues
Gloves
3. Preparation– If room temperature EDTA blood is being used the blood should be placed on the blood rocker for 10-15 minutes to allow adequate mixing of the blood cells. If the blood was refrigerated, mix the tube for at least 20 minutes.
4. Performance Parameters–N/A
5. Storage Requirements–Store at room temperature.
SPECIMEN
1. Patient Preparation–N/A
2. Type–Free-flowing capillary or thoroughly mixed anticoagulated venous blood. EDTA is the anticoagulant of choice.
Finger Stick
Refer to detailed description of this technique in Specimen Collection Section.
1. Put on gloves
2. Wipe the finger with alcohol and allow it to dry.
3. Make a puncture wound with a sterile lancet.
4. Check that the patient’s hand is relaxed. Some patients will hold their hand open, but it will be tense. This will prevent blood flow to the small capillaries and will prevent sample collection. It may be necessary to shake the patient’s hand to make it loose.
5. Do not squeeze the finger excessively. This radically alters the relationship between the plasma and the RBCs and will therefore change the micro hematocrit.
6. Wipe away the first drop of blood with a gauze pad.
7. Hold the capillary tube to the puncture site, tilting it in a downward direction. This will allow the tubes to fill much faster.
8. Fill the capillary tube at least three-quarters full, and insert the end of the tube into the clay sealant. Either end of the tube can be put into the sealant, but using the clean end will keep the sealant surface clean. If the other end is used, it should be wiped with a gauze to clean off excess blood from the outer surface of the tube.
9. Repeat the above steps with a second capillary tube.
Heel Stick
Refer to detailed description of the infant heel-stick technique in Specimen Collection Section.
EDTA-Anticoagulated Blood Tube
An accurate micro hematocrit can be obtained using EDTA-anticoagulated blood for up to 24 hours after its collection as long as the sample has been refrigerated. If left at room temperature it is good for only 6 hours.
1. Examine anticoagulated specimens carefully for clots. If the blood has clotted, the test cannot be accurately performed. Blood should be mixed well and at room temperature.
2. Put on gloves.
3. Hold a 2x2 inch gauze around the stopper and gently remove the stopper from the tube. Try to avoid splashing blood.
4. Put the micro hematocrit capillary tubes into the purple-top tube and collect the blood sample. Hold the tube as close to horizontal as possible without spilling the blood. Blood should rise into the tube by capillary action. If this does not happen, tap the top of the capillary tubes several times to speed up the process.
5. Wipe the blood off the outside of the capillary tube with gauze.
6. Place the capillary tubes into the sealant after they are at least three-fourths filled with blood. The more blood in the tube the easier the result is to read and the more accurate is the result.
QUALITY CONTROL
Test is performed in duplicate and results must agree within 2.0%. If not, repeat test.
Paired studies–Run a patient’s blood and send the patient’s sample to a reference laboratory for a complete blood count. The automated hematology equipment calculates the micro hematocrit from the red cell size and the red cell number. This calculated micro hematocrit value would be about 3% less than the micro hematocrit. This is due to the 3% plasma trapping that occurs in even the ideal micro hematocrit technique.
Participation in a proficiency testing survey.
CALIBRATION
It is necessary to perform periodic calibration of the timer (with a stopwatch) and of the centrifuge speed (rotations per minute, RPM). Speed may be calibrated using a tachometer device. If such a device is not available, the performance of the centrifuge can be checked by determining the “constant packing time.” Fill and seal a micro hematocrit tube and take a reading after 2 minutes of centrifugation. Centrifuge again for an additional minute and take a reading. Repeat centrifugation for another minute until there is no change in readings. The results should be recorded. For scheduled cleaning and maintenance, follow the manufacturer’s directions.
PROCEDURE
STANDARD PRECAUTION: Patient specimens and all materials coming into contact with them should be handled as if capable of transmitting infections and disposed of with proper precautions. Gloves should be worn when handling all specimens.
1. Place both capillary tubes in the centrifuge across from each other. The sealed end should be on the outside, as the centrifugal forces press the cells toward the outside of the centrifuge.
2. Place the lid on the centrifuge head. Most centrifuges have a special spin-on head to prevent the capillary tubes from flying out of the head during the centrifugation.
3. Centrifuge the capillary tubes for 5 minutes at a minimum of 15,000 g.
4. Remove the capillary tubes after spinning has stopped. To read the micro hematocrit, place the tube at the right edge of the reader card (see Figure 1) with the top of the plasma line at the 100% mark. Slide the tube to the left until the top of the clay sealant is at the bottom line (0). Check that the top of the plasma is at the 100% line, the bottom of the red cell column is at the 0 line, and the capillary tube is parallel to the edge of the reading card.
5. Carefully find the point where the top of the red cell column crosses the line on the reader card. Do not include the gray buffy coat just above the red cell column as part of the reading. The identified line corresponds to the micro hematocrit value. It may be necessary to use a magnifying glass to accurately identify the top of the red cell column.
6. Repeat the reading process for the second capillary tube. The results should agree within two percentage points. The reported value is the average of those values. If the two values differ by greater than 3 points, the entire procedure should be repeated.
INTERPRETATION
Examination of the micro hematocrit:
· Top of tube – fatty layer and normally barely visible. With lipemia, the layer is several mm thick.
· Second layer is plasma – pale yellow and fairly clear. Excessive hemolysis results in reddish color, and can lower the micro hematocrit. A new specimen should be obtained. Jaundice results in a yellow color.
· Third layer is buffy coat – 0.5 to 1.0 mm thick. If WBC count is over 10,000/cu mm, the layer is usually over 1 mm thick. Packed platelets are found in upper part of the layer and WBC in the lower part.
· Fourth layer is packed red cells – read as the micro hematocrit. A consistent technique for the micro hematocrit should produce a test-to-test variation of only + 2 percent. The largest source of error is the variation between readers in the reading step of the procedure.
FIGURE 1.
CALCULATIONS – N/A
REPORTING
Normal Values
The reference intervals based on Texas Department of Health (TDH) values are:
40-54% males
38-47% females
44-64% newborns
35-49% 14-90 days
30-40% 6 months – 1 year
31-43% 1 year – 10years
PROCEDURE NOTES
1. When collecting the fingertip blood, care should be taken not to squeeze the finger so as to contaminate the collection with tissue juice. This will decrease the micro hematocrit.
2. Capillary tubes should be centrifuged at 15,000g. A decreased centrifuged force will trap plasma between the RBC and cannot be corrected by increasing time. Inadequate centrifugation has stopped, will give falsely elevated readings. The time and speed of centrifugation, therefore, are extremely important in order to obtain maximal red cell packing.
3. An office laboratory may be 3% higher than the calculated hematocrit obtained from a reference laboratory. This is because of the small volume of plasma that is trapped between the red cells during the packing procedure.
4. Air bubbles that occur when filling the micro hematocrit tubes will not affect the final value because the bubbles will be expelled by the red cells as they migrate during the centrifugation process.
5. The microhematocrit is greatly affected by abnormally shaped cells because such cells trap plasma and artificially elevate the value of the micro hematocrit. Patients with distorted cells such as in sickle cell disease should therefore be followed by hemoglobin levels rather than microhematocrit values.
6. A good relationship to keep in mind is that the micro hematocrit is usually three times the value of the hemoglobin value. Another way to describe this relationship is that 1 hematocrit point is equivalent to 0.34 g Hb/dL.
SOURCES OF ERROR
Falsely Low Values
1. Improper reading of the micro hematocrit value from the reader chart
2. Inadequate mixing of an EDTA-anticoagulated tube and drawing of the specimen from the plasma-rich area at the top of the tube
3. Inadequately filling an EDTA tube with blood so that the anticoagulant over dilutes the blood sample
4. Reading the micro hematocrit a long period after the sample has been spun
Falsely High Values
1. Incorrect reading of the micro hematocrit from the reader chart
2. Inclusion of the buffy coat in the reading of the red cell column
3. Inadequate mixing of an EDTA-anticoagulated tube and drawing of the specimen from the cell-rich area at the bottom of the tube
4. Inadequate centrifuge speed or time, resulting in less than maximal packing of the red blood cells
5. Increased plasma trapping because of unusually shaped cells (e.g., macrocytes, sickle cells, poikilocytosis). This can result in as great an increase as 20 percent in extreme cases, such as sickled cells.
6. The use of EDTA-anticoagulated blood that has been stored for greater than 24 hours
REFERENCES
1. Clinical Diagnostics & Management, 18th Ed., Mar 1995, Pg. 583 by J.B. Henry, M.D.
2. Laboratory Test Handbook, 3rd Ed. With Key Word index, 1994, Pg. 578, 581, and 582-583 by Jacobs, Demott, Finley, Horvatm Kasten, Tilzer
3. National Committee for Clinical Laboratory Standards (NCCLS), Physician’s Office Laboratory Guidelines, 2nd ed. Document POL1-T2. Villanova, PA: June, 1995
The micro hematocrit represents the volume of circulating blood that is occupied by erythrocytes and is expressed as a percentage. The micro hematocrit test helps the medical provider identify patients at risk of developing anemia and manage patients who have anemia. Anemia is a less than normal number of erythrocytes in the volume of packed cells. The most common cause of anemia throughout the world is iron deficiency. A decrease in the micro hematocrit value results in a lack of energy, a reduced resistance to infections, and an increased risk of abnormality in pregnancy. An increased micro hematocrit value can result from polycythemia, hemoconcentration, and/or cardiac disease.
SUPPLIES AND EQUIPMENT
1. Equipment
High-speed centrifuge
Micro hematocrit tube reader
Timer
2. Materials/Reagents
Capillary tubes (heparinized) if finger stick specimens used
Capillary tubes (nonheparinized) if whole blood is used
Clay to seal tubes
Laboratory tissues
Gloves
3. Preparation– If room temperature EDTA blood is being used the blood should be placed on the blood rocker for 10-15 minutes to allow adequate mixing of the blood cells. If the blood was refrigerated, mix the tube for at least 20 minutes.
4. Performance Parameters–N/A
5. Storage Requirements–Store at room temperature.
SPECIMEN
1. Patient Preparation–N/A
2. Type–Free-flowing capillary or thoroughly mixed anticoagulated venous blood. EDTA is the anticoagulant of choice.
Finger Stick
Refer to detailed description of this technique in Specimen Collection Section.
1. Put on gloves
2. Wipe the finger with alcohol and allow it to dry.
3. Make a puncture wound with a sterile lancet.
4. Check that the patient’s hand is relaxed. Some patients will hold their hand open, but it will be tense. This will prevent blood flow to the small capillaries and will prevent sample collection. It may be necessary to shake the patient’s hand to make it loose.
5. Do not squeeze the finger excessively. This radically alters the relationship between the plasma and the RBCs and will therefore change the micro hematocrit.
6. Wipe away the first drop of blood with a gauze pad.
7. Hold the capillary tube to the puncture site, tilting it in a downward direction. This will allow the tubes to fill much faster.
8. Fill the capillary tube at least three-quarters full, and insert the end of the tube into the clay sealant. Either end of the tube can be put into the sealant, but using the clean end will keep the sealant surface clean. If the other end is used, it should be wiped with a gauze to clean off excess blood from the outer surface of the tube.
9. Repeat the above steps with a second capillary tube.
Heel Stick
Refer to detailed description of the infant heel-stick technique in Specimen Collection Section.
EDTA-Anticoagulated Blood Tube
An accurate micro hematocrit can be obtained using EDTA-anticoagulated blood for up to 24 hours after its collection as long as the sample has been refrigerated. If left at room temperature it is good for only 6 hours.
1. Examine anticoagulated specimens carefully for clots. If the blood has clotted, the test cannot be accurately performed. Blood should be mixed well and at room temperature.
2. Put on gloves.
3. Hold a 2x2 inch gauze around the stopper and gently remove the stopper from the tube. Try to avoid splashing blood.
4. Put the micro hematocrit capillary tubes into the purple-top tube and collect the blood sample. Hold the tube as close to horizontal as possible without spilling the blood. Blood should rise into the tube by capillary action. If this does not happen, tap the top of the capillary tubes several times to speed up the process.
5. Wipe the blood off the outside of the capillary tube with gauze.
6. Place the capillary tubes into the sealant after they are at least three-fourths filled with blood. The more blood in the tube the easier the result is to read and the more accurate is the result.
QUALITY CONTROL
Test is performed in duplicate and results must agree within 2.0%. If not, repeat test.
Paired studies–Run a patient’s blood and send the patient’s sample to a reference laboratory for a complete blood count. The automated hematology equipment calculates the micro hematocrit from the red cell size and the red cell number. This calculated micro hematocrit value would be about 3% less than the micro hematocrit. This is due to the 3% plasma trapping that occurs in even the ideal micro hematocrit technique.
Participation in a proficiency testing survey.
CALIBRATION
It is necessary to perform periodic calibration of the timer (with a stopwatch) and of the centrifuge speed (rotations per minute, RPM). Speed may be calibrated using a tachometer device. If such a device is not available, the performance of the centrifuge can be checked by determining the “constant packing time.” Fill and seal a micro hematocrit tube and take a reading after 2 minutes of centrifugation. Centrifuge again for an additional minute and take a reading. Repeat centrifugation for another minute until there is no change in readings. The results should be recorded. For scheduled cleaning and maintenance, follow the manufacturer’s directions.
PROCEDURE
STANDARD PRECAUTION: Patient specimens and all materials coming into contact with them should be handled as if capable of transmitting infections and disposed of with proper precautions. Gloves should be worn when handling all specimens.
1. Place both capillary tubes in the centrifuge across from each other. The sealed end should be on the outside, as the centrifugal forces press the cells toward the outside of the centrifuge.
2. Place the lid on the centrifuge head. Most centrifuges have a special spin-on head to prevent the capillary tubes from flying out of the head during the centrifugation.
3. Centrifuge the capillary tubes for 5 minutes at a minimum of 15,000 g.
4. Remove the capillary tubes after spinning has stopped. To read the micro hematocrit, place the tube at the right edge of the reader card (see Figure 1) with the top of the plasma line at the 100% mark. Slide the tube to the left until the top of the clay sealant is at the bottom line (0). Check that the top of the plasma is at the 100% line, the bottom of the red cell column is at the 0 line, and the capillary tube is parallel to the edge of the reading card.
5. Carefully find the point where the top of the red cell column crosses the line on the reader card. Do not include the gray buffy coat just above the red cell column as part of the reading. The identified line corresponds to the micro hematocrit value. It may be necessary to use a magnifying glass to accurately identify the top of the red cell column.
6. Repeat the reading process for the second capillary tube. The results should agree within two percentage points. The reported value is the average of those values. If the two values differ by greater than 3 points, the entire procedure should be repeated.
INTERPRETATION
Examination of the micro hematocrit:
· Top of tube – fatty layer and normally barely visible. With lipemia, the layer is several mm thick.
· Second layer is plasma – pale yellow and fairly clear. Excessive hemolysis results in reddish color, and can lower the micro hematocrit. A new specimen should be obtained. Jaundice results in a yellow color.
· Third layer is buffy coat – 0.5 to 1.0 mm thick. If WBC count is over 10,000/cu mm, the layer is usually over 1 mm thick. Packed platelets are found in upper part of the layer and WBC in the lower part.
· Fourth layer is packed red cells – read as the micro hematocrit. A consistent technique for the micro hematocrit should produce a test-to-test variation of only + 2 percent. The largest source of error is the variation between readers in the reading step of the procedure.
FIGURE 1.
CALCULATIONS – N/A
REPORTING
Normal Values
The reference intervals based on Texas Department of Health (TDH) values are:
40-54% males
38-47% females
44-64% newborns
35-49% 14-90 days
30-40% 6 months – 1 year
31-43% 1 year – 10years
PROCEDURE NOTES
1. When collecting the fingertip blood, care should be taken not to squeeze the finger so as to contaminate the collection with tissue juice. This will decrease the micro hematocrit.
2. Capillary tubes should be centrifuged at 15,000g. A decreased centrifuged force will trap plasma between the RBC and cannot be corrected by increasing time. Inadequate centrifugation has stopped, will give falsely elevated readings. The time and speed of centrifugation, therefore, are extremely important in order to obtain maximal red cell packing.
3. An office laboratory may be 3% higher than the calculated hematocrit obtained from a reference laboratory. This is because of the small volume of plasma that is trapped between the red cells during the packing procedure.
4. Air bubbles that occur when filling the micro hematocrit tubes will not affect the final value because the bubbles will be expelled by the red cells as they migrate during the centrifugation process.
5. The microhematocrit is greatly affected by abnormally shaped cells because such cells trap plasma and artificially elevate the value of the micro hematocrit. Patients with distorted cells such as in sickle cell disease should therefore be followed by hemoglobin levels rather than microhematocrit values.
6. A good relationship to keep in mind is that the micro hematocrit is usually three times the value of the hemoglobin value. Another way to describe this relationship is that 1 hematocrit point is equivalent to 0.34 g Hb/dL.
SOURCES OF ERROR
Falsely Low Values
1. Improper reading of the micro hematocrit value from the reader chart
2. Inadequate mixing of an EDTA-anticoagulated tube and drawing of the specimen from the plasma-rich area at the top of the tube
3. Inadequately filling an EDTA tube with blood so that the anticoagulant over dilutes the blood sample
4. Reading the micro hematocrit a long period after the sample has been spun
Falsely High Values
1. Incorrect reading of the micro hematocrit from the reader chart
2. Inclusion of the buffy coat in the reading of the red cell column
3. Inadequate mixing of an EDTA-anticoagulated tube and drawing of the specimen from the cell-rich area at the bottom of the tube
4. Inadequate centrifuge speed or time, resulting in less than maximal packing of the red blood cells
5. Increased plasma trapping because of unusually shaped cells (e.g., macrocytes, sickle cells, poikilocytosis). This can result in as great an increase as 20 percent in extreme cases, such as sickled cells.
6. The use of EDTA-anticoagulated blood that has been stored for greater than 24 hours
REFERENCES
1. Clinical Diagnostics & Management, 18th Ed., Mar 1995, Pg. 583 by J.B. Henry, M.D.
2. Laboratory Test Handbook, 3rd Ed. With Key Word index, 1994, Pg. 578, 581, and 582-583 by Jacobs, Demott, Finley, Horvatm Kasten, Tilzer
3. National Committee for Clinical Laboratory Standards (NCCLS), Physician’s Office Laboratory Guidelines, 2nd ed. Document POL1-T2. Villanova, PA: June, 1995
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