Manual Cell Count and Differential For Body Fluid and CSF

Manual Cell Count for Body Fluid and CSF [HHM.
PROC-53672]
Mayo Clinic Laboratories

Mayo Clinic Health System (MCHS)
Hematology
Manual Cell Count and Differential for Body Fluid and CSF
CONTENT CLASSIFICATION
Mark all that apply: Analytical Pre-Analytical Post-Analytical Non-Analytical
SCOPE
This document applies to the following MCHS location(s):
 MCHS-NW WI Region applies to Barron, Eau Claire Hospital, Menomonie
 MCHS-SE MN Region, River Corridor applies to Red Wing
 MCHS-SE MN Region, I90-I35 Corridor applies to Albert Lea, Austin
 MCHS-SW MN Region applies to Fairmont, Mankato, New Prague Hospital
 MCHS-SW WI Region applies to La Crosse Campus
INDEX
Specimens Reagents/Supplies/Equipment Quality Control Procedure Calculations
Reporting Results Procedural Notes References Appendix A Appendix B Attach 1 Attach 2
PURPOSE
This procedure is designed to provide instructions for performing a manual cell count and nucleated cell
differential in body fluids and cerebrospinal fluid (CSF) samples.
PRINCIPLE
 Cerebrospinal Fluid (CSF): The product of continuous secretory activity of the choroid plexuses of the
brain. The fluid acts as a protective cushion for the underlying central nervous system tissue. To detect by
means of observation, cell count and 100 nucleated cell differential any abnormalities in CSF; this may be
diagnostic for organic central nervous system disease or intracranial hemorrhage.
 Body Fluid (BF): The closed cavities of the body - the pleural, pericardial, and peritoneal cavities - are each
lined by two membranes referred to as the serous membranes. One membrane lines the cavity wall (parietal
membrane), and the other covers the organs within the cavity (visceral membrane). The fluid between the
membranes, which provides lubrication as the surfaces move against each other, is called serous fluid.
Fluids for cell count are divided into two categories: transudates or exudates. An abnormal accumulation
may occur in systemic disease, inflammation, malignancy, infection, and trauma.
 Normal synovial fluid, often referred to as “joint fluid”, is a viscous liquid found in the joint cavities having
essentially the same chemical composition as the plasma. Analysis of synovial fluid cell count is used to
determine classification and pathologic significance of joint disorders.
 Bronchoalveolar lavage (BAL) is a technique used to obtain samples of cellular material from the
pulmonary alveoli. It is performed during bronchoscopy by lavaging a sub-segment of lung with aliquots of
normal saline and collecting this fluid under low suction. Analysis of BAL fluid can confirm the presence
of a variety of bacterial, viral, fungal, and parasitic infections.
SPECIMENS Index
All fluids collected should be sent to the laboratory as soon as possible after collection. Samples must be tested
immediately upon receipt in the lab because WBCs (particularly granulocytes) and RBCs will begin to lyse
within 1 hour, with 40% of the leukocytes disintegrating after 2 hours. Patient preparation will be the
responsibility of the provider collecting the specimen.
Master copies are retained online. Printed copies are considered current only on the date printed unless stamped Controlled. Page 1 of 14
HHM.PROC-53672: 10.0 (EFFECTIVE Jul 27 2023 1:00AM) Printed On: Jul 27 2023 7:21AM dbMCValidation
Manual Cell Count for Body Fluid and CSF [HHM.PROC-53672]
 See Attachment 1 for acceptability requirements for body fluid counts and differentials.
 All samples submitted for a Cell Count must be collected in an EDTA tube with the exception of CSF.
 Samples must be labeled with the patient’s name, one more unique identifier MRN preferred, site, and
source.
 Body fluid specimens should never be transported via pneumatic tube system.
 If the sample received is ≤500 mcL, a manual cell count and differential will be attempted but not
guaranteed. Contact the provider to determine which test is most important if the quantity is limited.
 CSF samples will be retained refrigerated for a minimum of one month. All other body fluid samples will
be retained refrigerated for a minimum of seven days.
 Peritoneal Dialysate Fluid: While technically not designated as body fluid, peritoneal dialysates are received
for laboratory analysis from patients on peritoneal dialysis.
 CSF: The amount of sample obtained varies, but approximately 1.0 mL should be submitted for cell count
and differential. Tubes should be distributed for testing as follows, unless the provider requests testing to be
performed on other tubes:
Tube #1 Chemistry
Tube #2 Microbiology
Tube #3 Hematology Cell Count (if last tube collected)
Tube #4 Hematology Cell Count (if last tube collected), Cytology, or other Misc. Testing
NOTE: Cell counts should always be done on the last tube collected unless the physician requests an
additional cell count done on another tube or a traumatic collection is suspected. Blood from a cerebral
hemorrhage will be evenly distributed throughout all tubes collected; whereas a traumatic tap will have the
heaviest concentration of blood in tube #1, with gradually diminishing amounts in subsequent tubes.
REAGENTS/SUPPLIES/EQUIPMENT Index
 Hyaluronidase  Cytocentrifuge
 Test tube with cap  Lab-specific staining methodology
 Clean microscope slide – Charged slide  Pipette tips
preferred  Saline
 Dispo-pipette  Gloves
 Microscope with phase contrast capability  Timer
 Calibrated MLA pipette  Biological safety cabinet (for BAL samples)
 C-Chip Disposable Hemocytometer - System Neubauer Improved
QUALITY CONTROL Index

1. Streck Cell Chex: Level 1 (normal) and Level 2 (abnormal): At least one manual cell count control must be
performed once per technologist/8-hour shift if manual counts are performed on patients. Control levels are
analyzed on an alternating basis. Cell-Chex is an assayed control intended for monitoring total cell counts
performed manually using a hemocytometer to validate quantitation of red and white blood cells in patient
cerebrospinal fluid and body fluid samples including pleural, pericardial, peritoneal, and synovial fluid.
NOTE: Cell-Chex Level one may contain stabilized crystals which may appear as precipitate when
performing manual cell count quality control and can be ignored.
2. Refer to Appendix A and/or Appendix B for quality control ad hoc directions.
3. When Cell-Chex is not opened, it is stable through the expiration date when stored at 2-10°C. After
opening, Cell-Chex is stable for 30 days, as indicated on the manufacturer's assay sheet, when stored at
2-10°C. Discard the controls if they are empty or there is any evidence of microbial contamination.
4. Remove the choice of control from the refrigerator. Check the open vial dating expiry. It is not necessary
to warm the controls to room temperature before using.
5. To mix (do not mix mechanically or vortex):

a. Hold vial vertically and roll each vial between the palms of the hands for 15-20 seconds.
b. Continue to mix by holding the vial by the ends between the thumb and finger, rapidly inverting the vial
20 times end-over-end using a very quick turning motion of the wrist.
c. Analyze immediately after mixing. Subsequent analyses during the test period may be performed by
inverting the vial 5 times prior to sampling.
d. Steps a-c must be repeated upon removing the sample from the refrigerator for the entire open-vial time
period.
e. Thorough mixing with each use is critical in order to achieve precision, the degree of
reproducibility of results.
6. Open the pack of the C-Chip (DHC-N01) Disposable Hemocytometer – System Neubauer Improved. Make
sure it is clean and dry. The C-Chip is for single use only and must be used immediately after unsealing.
(The warranty on the C-Chip included in the conditions of supply is valid for 36 months from the date of
manufacturing.)
7. Using a certified 10 mcL MLA pipette and clean pipette tips, load 10mcL of the sample into the sample
injection port in Figure 2. The chamber should fill by capillary action. Care should be taken to prevent
cross-contamination of the control. Do not underfill or overfill. Ensure enough fluid is introduced so that
the surface of the counting area is just covered. Label the hemocytometer so that the sample can be properly
identified.
8. After sampling, return Cell-Chex to refrigeration for maximum open-vial stability. Wipe the threads of both
the vial and cap before replacing cap and returning to refrigeration.
9. Count the total nucleated cells (TNCs) and red blood cells (RBCs) in all 9 large squares on both chamber
sides of the C-Chip Neubauer hemocytometer as seen in the Figure 1.
10. Average the number of cells (TNCs and RBCs, respectively) counted on both sides.
a. If <100 cells are counted on each chamber side, the two chamber counts must match within ±10 cells.
b. If >100 cells are counted on each chamber side, the two chamber counts must match within ±20%.
11. Calculate the total number of each TNCs and RBCs per mcL (see worksheet).
12. Document the results on the Body Fluid/CSF Worksheet [HHM.F-63604].
13. Log QC results within the TQC management system.
14. If control values are not in range:
a. Verify open/expiration date of controls.
b. Perform a second count on the same level of QC.
c. Obtain a fresh vial of the same level of QC and perform a count.
d. Another tech should perform the count to verify (if possible).
e. If failure persists, hold patient results and contact the supervisor, TS or pathologist.
15. New Control Lot Assessment/Studies:
a. Performance of quality control materials and evaluation of manufacturer’s assay ranges are assessed
prior to using a new lot of controls.
b. When preparing to start a new lot, both levels will be assessed one time for acceptability to validate the
manufacturer range and documented in TQC prior to patient testing.
c. New lot QC sample preparation is performed according to manufacturer package insert.
PROFICIENCY TESTING
1. Refer to Proficiency Testing and Alternative Assessment Policy [HQ.POL-64864]
2. All staff performing body fluid cell counts will participate in annual competency assessment.
PROCEDURE Index
Step Action
1. Fill out the Body Fluid/CSF Worksheet [HHM.F-63604] for each specimen.
a. Place an LIS label or complete patient identification information on each worksheet.
b. Document the fluid type and the site if applicable. (i.e., left, right, upper, lower, knee, elbow, etc.)
c. Document appearance and color of the fluid
d. Document if Cytology was ordered.
e. If a Cytology is ordered on a fluid and the fluid must be transported to another site or the
Histology lab is not staffed, an aliquot of sample must be refrigerated.
f. Document if the fluid needed a dilution or not. If the sample was diluted write down the dilution
factor used.
2. Xanthochromia (Must be performed on all CSF specimens for cell count)
a. If CSF is clear and colorless, xanthochromia will be resulted as negative.
b. If the specimen appears bloody or has an orange or yellowish tint, centrifuge to determine the
color of the supernate.
1) Pipette 500-1000 mcL of CSF into an aliquot tube. A minimum of 200 mcL is needed to
perform the test.
Step Action
2) Cover aliquot tube and centrifuge for 5 minutes at 3500 RPM.
3) Hold the centrifuged CSF up against a white background and compare to the water blank.
4) Report as negative (colorless); positive (pink, orange, yellow); unable to determine (red or
another color or interference)
3. Manual Cell Count:
a. Mix sample well.
b. Charge a C-Chip disposable hemocytometer with 10 mcL of well-mixed undiluted fluid sample
into the sample injection area on both sides of the hemocytometer slide. Use samples as follows:
1) CSF: Sample should be well mixed and undiluted. Anticoagulants should never be added to
CSF.
2) Joint fluid: Mix EDTA anticoagulated sample well. If specimen is viscous use a wooden
applicator stick to dispense a “pinch” of hyaluronidase (approximately 5 mg) hyaluronidase to
a 1.0 mL aliquot of fluid. Gently mix well, let sit for 5 minutes, and mix well again.
NOTE: Perform crystal analysis before adding hyaluronidase.
3) Serous fluid: Well mixed EDTA anticoagulated sample
c. If a pellicle (yellowish clump) is present, document on the worksheet “Count approximate due to
pellicle” (@HQ71). If a clot is present, document on the worksheet “Count approximate due to clot.”
(@HQ70).
d. Scan the large squares of the hemocytometer using low power (10X) for an estimated count.
Look for an even distribution of cells without overlap or clumping of the cells; if cells overlap a
dilution must be made.
NOTES:
 The RBC dilution could be different than the nucleated cell dilution depending on how many
cells were initially seen under the scope.
 Samples can be diluted with isotonic saline or Sysmex Cellpack reagent for both white and
red cell dilutions.
e. If a dilution is necessary, visually examine your diluent by placing a small amount of the diluting
fluid on a microscope glass slide, placing a coverslip on the slide, and visually examining the
diluent for non-specimen background particulates. If particles are seen, obtain a fresh aliquot of
diluent. Document on worksheet.
f. Use the certified and calibrated MLA pipettes in a clean plastic tube or sterile urine cup to make a
sample dilution.
NOTE: Do not over-dilute the specimen. You may not report zero (0) for any cell type if
performing on a dilution.
Dilution Patient Cellpack
Dilute 1:2 200 mcL 200 mcL
Step Action
g. If numerous RBCs obscure the TNCs or are difficult to differentiate from the TNCs, rinse the
pipette tip with glacial acetic acid to lyse the RBCs and pipette an aliquot of sample to load into
the hemocytometer chambers.
NOTE: Acetic acid is not suitable for use with synovial fluids.
h. Count the Total Nucleated Cells (TNC) using the 40X objective. The TNCs should have a
nucleus that is visible or may appear a little grainy. Most TNCs are larger when compared to the
RBCs but keep in mind that the lymphocytes can be similar in size. Macrophages/Monocytes
may appear much larger. All cells with a nucleus will be counted towards the Total Nucleated
Count.
i. CSF ONLY: Count the RBC using the 40X objective.
All cell counts should be performed as follows:
Cell Estimate or dilution Number of Squares to Count Square Area
 Less than 200 cells present in all Count all 9 nine squares Area is equal to 9 mm2
nine squares or
 No dilution or
 1:2, 1:5, or 1:10 dilution
 More than 200 cells present in all Count 4 corner squares: see Area is equal to 4 mm2
nine squares or figure 1 (1,3,7,9)
 1:21, 1:41, or 1:101 dilution
 More than 200 cells present in one Count 5 squares within the Area is equal to 0.2 mm2
square or center square: see figure 2,
 1:201 dilution dark shaded areas.
Figure 1 Figure 2
j. Repeat count on second chamber.
1) If <100 cells are counted on each chamber side, the two chamber counts must match within
± 10 cells.
2) If >100 cells are counted on each chamber side, the two chamber counts must match within
± 20%.
k. Calculate the average number of each TNCs and RBCs per mcL as follows:
 Average # cells counted x dilution factor (if used) = Cells/mcL
2
Average Area (mm ) counted x Chamber Depth (0.1)
NOTE: If no cells are seen on hemocytometer, report count as <1. Continue to attempt cytospin
Step Action
differential.
l. Prepare a cytospin slide according to local process and count a 100-cell differential.
1) For synovial fluid, the aliquot of sample treated with hyaluronidase may be used to make the
cytospin slide.
2) Scan the entire slide for cell distribution and for the presence of cell clumps which can
indicate malignancy. Ensure that the edges of the smear are examined.
3) Ensure uniform cell distribution, appropriate dilution so cells are not crowded, proper
staining, adequate cell yield, and ready recognition of cell types that are reported.
4) Perform a 100-cell differential unless cell recovery does not allow for 100 cells to be
differentiated. Count the identifiable cells. Consider preparing additional smears to aid in
cell recovery.
5) If less than 10 cells are seen after examination of additional smears, add comment “Too few
white cells for accurate differential”. In Soft under comment, select See Comment, select
comment, and report @HP06.
6) If abnormal cells are seen, refer to Attachment 2 for identification guidance.
m. Document the results on the Body Fluid/CSF Worksheet [HHM.F-63604].
n. Have a second tech review the calculations on the Body Fluid Worksheet and sign off. If you are
alone a review of the math should be completed as soon as someone is available to do the review.
o. Enter the results into Caresphere/LIS appropriately.
p. Site-specific completion instructions.
SWMN/SEMN SWWI NWWI/River Corridor
Send the completed When atypical cells or If any abnormal cells are seen – contact
worksheet and the cells suspicious for Eau Claire Hospital. Please send an
slides to Mankato for malignancy are observed aliquot of the specimen, slide, and copy of
pathologist review. on the differential slide BF/CSF worksheet to Eau Claire Hospital.
Enter the pathologist’s preparation, the slide and ECH techs- consult Cytology.
comments in the completed form should 1. Bring sample and Wright stain (diff)
patient’s record when be submitted to a slide into cytology.
they are returned from cytologist or pathologist 2. Place paperwork and diff slide under
Pathology or amend the for review prior to final hood in cytology.
report for any result reporting. 3. Place sample in refrigerator #31 in the
comments returned on red bin labeled New Specimens.
slides referred for 4. Cytology- make a Pap-stained cytospin
review. slide.
a. Coverslip both slides.
b. Take paperwork and slide to
pathologists.
CALCULATIONS Index
NOTE: Perform all calculations on the Body Fluid Worksheet [HHM.F.63604]
 The following formulas used to calculate the cell count:
Cells/mcL = Total # cells counted x dilution factor (if used)
Total Number of mm2 counted x Chamber Depth (0.1)
Example:
o RBCs counted = 150
Nucleated cells counted = 175
Dilution: No dilution
Estimate was for all 9 squares – all 9 squares were counted.
REPORTING/INTERPRETING RESULTS Index

1. Reference range: See Reference Ranges: Hematology and Coagulation Testing [HHM.QRG.61062].
2. Caresphere sites: refer to Sysmex Caresphere Workflow Solution (CWS) [HHM.PROC-64315] to report
the manual cell count and differential results.
3. Non-Caresphere sites: Reporting Results
a. Select the appropriate Resulting Worklist in Soft.
b. Result your specimen by reporting all tests listed. See screenshot below.
c. Use Diffpad to perform differential count. Count can be changed by selecting the Change Diffpad Limit.
Verify count.
d. Result the Reviewed by box with your Soft tech code.
e. Verify and Save.
REFERENCES Index
Document/Form Title
Galagan KA, et al: CAP Hematology and Clinical Microscopy Resource Committee. (2006) Color Atlas of
Body Fluids: An Illustrated Field Guide Based on Proficiency Testing. Northfield, IL: College of American
Pathologists.
Kjeldsberg CR, etal: Body Fluid Third Edition: Laboratory Examination of Amniotic, Cerebrospinal, Seminal,
Serous & Synovial Fluids. (1993) Hong Kong: The American Society of Clinical Pathologists.
Mayo Clinic: Department of Laboratory Medicine and Pathology; Division of Hematopathology,
Hematopathology Morphology Laboratory; Rochester, MN: Body Fluid Cell Count for Synovial and Other
Body Fluids [007721].
Mayo Clinic: Department of Laboratory Medicine and Pathology; Division of Hematopathology,
Hematopathology Morphology Laboratory; Rochester, MN: CSF, Ventricular Fluid, Vitreous Fluid, Flap
Fluid and Lavage Cell Count [007723].
Elements considered but determined to be not applicable for this procedure are: Definitions/Acronyms,
Equipment, Calibration, Troubleshooting, Procedural Notes, Limitations, Related Documents, Related Training
Appendix A: Index
Manual Body Fluid or CSF Cell Count – TQC – TECH HAS NO CURRENT QC
(You have not performed a manual cell count control within the past 8 hours.)
1. You will need to have active lots in TQC for: BFMAN_1 and BFMAN_2
2. You have received an order for a Body Fluid and determined that you cannot perform testing using an
automated method, or the automated cell count obtained is less than the acceptable background count.
You have not performed manual cell count QC within the past 8 hours.
3. In Resulting Worklist, locate your body fluid order.
4. Right Click on the ‘Fluid Type’ field and choose Add QC Order. The following screen will appear:
5. Fill in the Location, Item Type, and Department. Click “Find”

A. Location – Choose applicable site
B. Item Type – Control
C. Department – Choose applicable department
6. The following screen will appear:
7. Check the BFMAN control you will need to count.

A. BFMAN_1 = (L1)
B. BFMAN_2 = (L2)
8. Click “OK.”
9. The following screen will appear. Select the controls you will result and click on “Generate Orders” in the
lower right-hand corner.
10. If another operator has counted a control earlier in the day, you will see the following:
11. You will be bridged to TQC. Select “Open” and your control(s) will be ready to result.
12. Either print a barcode (Using the Print Labels icon) or record the QC order number for your controls. These
will be needed should you count another fluid later in your shift.
13. Enter your control results.
14. Return to Caresphere to enter your patient results (or SoftLab if Caresphere is unavailable).
15. Verify and Save.
Appendix B: Index
Manual Body Fluid or CSF Cell Count – TQC – TECH HAS CURRENT QC
(You have performed a manual cell count control within the past 8 hours)
1. You will need to have active lots in TQC for: BFMAN_1 and BFMAN_2 and BFMAN_P
2. You have received an order for a Body Fluid and determined that you cannot perform testing using an
automated method, or the automated cell count obtained is less than the acceptable background count.
You have performed manual cell count QC within the past 8 hours.
3. In Resulting Worklist, locate your body fluid order.
4. Right Click on the ‘Fluid Type’ field and choose Add QC Order. The following screen will appear:
5. Fill in the Location, Item Type, and Department. Click “Find”

A. Location – Choose applicable site
B. Item Type – Control
C. Department – Choose applicable department
6. The following screen will appear:
7. Check the BFMAN_P control. Click “OK.”

8. The following screen will appear.
9. Click on ‘Save’ in the upper left-hand corner.

10. If another operator has counted a control earlier in the day, you will see the following:
11. Choose ‘Yes’ to generate a new order.

12. You will be bridged to TQC and your control(s) will be ready to result.
13. Because you have counted a control within the past 8 hours, enter the QC identifier for the previously
counted control.
14. Recording the previously counted control number will allow a surveyor to relate your first QC of the shift to
this patient.
15. Verify and Save.
16. Return to Caresphere to enter your patient results (or SoftLab if Caresphere is unavailable).
Attachment 1 Index
Abnormal Specimens for Body Fluid Cell Counts and Differentials
Body Fluid Received Recommendation

 Pleural Fluid  Perform ordered tests according to current process (automated
 Peritoneal/Ascites Fluid cell count/manual cell count when indicated and differential).
 Pericardial Fluid
 Synovial Fluid
 Cerebrospinal Fluid
 Fluid from abscess source  Do not perform cell count (.TNP). Add comment, “Cell count
not performed due to source.”
 Consult with ordering provider regarding the necessity of
differential. If clinically indicated, perform differential.
 Recommend Gram stain/Culture and Cytology.
 Fluid from other “abnormal” source  Do not perform cell count (.TNP). Add comment, “Cell count
 Examples (but not limited to): and differential not performed due to abnormal source. See
o Bile pathologist interpretation.”
o Unknown fluid leaking into  Make and stain slide, but do not perform manual differential.
abdominal cavity Send slide and internal review form to pathology for review
o Gall Bladder Cysts during normal working hours.
o Gall Bladder Aspirates  Consult with ordering provider and recommend Gram
o Kidney Aspirate stain/Culture, and possible Cytology if clinically indicated.
o Ear Aspirate
o Breast fluid Aspirate
o Pelvic Cyst
 Bronchioalveolar Lavage (BAL)  Do not perform cell count (.TNP). Add comment, “Cell count
not performed due to source.”
 Perform differential.
Attachment 2 Index
Characteristics of Atypical Cells in Body Fluid Specimens

Clumping Examine on low power for the presence of three-dimensional clusters:
Mesothelial Atypical
Periphery of clump ‘knobby’ smooth border
Nucleus (position) central eccentric
Dual Population Two distinct populations of epithelial cells: mesothelial vs. other. Rule out spectrum.
N:C Ratio High; not all atypical cells will have very high N:C ratios.
Nucleoli +/-, prominent, may be irregular, often solitary vs. reactive (multiple, small)
Atypical Mitosis Atypical or bizarre mitoses are highly suspicious for malignancy.
Monotony Atypical population
Signet Ring Confirm dual epithelial population: if seen absent dual population these are most likely
cells macrophages. In the presence of dual epithelial population: suspicious, may contain mucin.
Notes  Clumping may be an artifact, especially in very thick preparations.
 Normal mesothelial and plasma cells may be multinucleated.
 Compare suspicious cells to cells that are easily recognized. Note the
difference/similarities in size, N:C ratio, nucleoli, and chromatin clumping.
 Consider that different cells may be opposing ends of a spectrum of normal variation.

Manual Cell Count and Differential For Body Fluid and CSF

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Manual Cell Count for Body Fluid and CSF [HHM.

Mayo Clinic Laboratories

QUALITY CONTROL Index

5. To mix (do not mix mechanically or vortex):

REPORTING/INTERPRETING RESULTS Index

5. Fill in the Location, Item Type, and Department. Click “Find”

7. Check the BFMAN control you will need to count.

5. Fill in the Location, Item Type, and Department. Click “Find”

7. Check the BFMAN_P control. Click “OK.”

9. Click on ‘Save’ in the upper left-hand corner.

11. Choose ‘Yes’ to generate a new order.

Abnormal Specimens for Body Fluid Cell Counts and Differentials

Body Fluid Received Recommendation

Characteristics of Atypical Cells in Body Fluid Specimens

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