Cross-matchingCross-matching blood, in transfusion medicine, refers to the complex testing that is performed prior to a blood transfusion, to determine if the donor's blood is compatible with the blood of an intended recipient, or to identify matches for organ transplants. Cross-matching is usually performed only after other, less complex tests have not excluded compatibility. Blood compatibility has many aspects, and is determined not only by the blood types (O,A,B,AB), but also by blood factors, (Rh, Kell, etc.)

Cross-matching is done by a certified laboratory technologist, in a laboratory. It can be done electronically, with a computer database, or serologically. Simpler tests may be used to determine blood type (only), or to screen for antibodies (only). (indirect Coombs test).

Contents

1 Types of cross-matching o 1.1 Electronic cross-matching o 1.2 Serological cross-matching

2 Emergencies

Types of cross-matching Electronic cross-matching

Electronic cross-matching is essentially a computer-assisted analysis of the data entered from testing done on the donor unit and blood samples drawn from intended recipient. This includes ABO/Rh typing of the unit and of the recipient, and an antibody screen of the recipient. Electronic cross-matching can only be used if a patient has a negative antibody screen, which means that they do not have any active red blood cell atypical antibodies, or they are below the detectable level of current testing methods. If all of the data entered is compatible, the computer will print a compatibility label stating that the unit is safe to transfuse.

Serological cross-matching

In serological cross-matching, red blood cells from the donor unit are tested against the plasma of the patient in need of the blood transfusion. If the patient’s serum contains antibodies against the antigens present on the donor red blood cells, agglutination will occur. Agglutination is considered a positive reaction indicating that the donor unit is incompatible for that specific patient. If no agglutination occurs the unit is deemed compatible and is safe to transfuse.

Emergencies

In the case of an emergency a physician can request "uncross-matched blood", or donor units of blood that have not been cross-matched. It is thought that this lifesaving measure is of more benefit than any risk of an antibody-mediated transfusion reaction. In addition, the risk of a serious transfusion reaction can be minimized if the donor unit is both ABO-compatible and Rhesus (Rh)-compatible. Type O and Rh negative blood can be given if the recipient's blood group is not known, as may happen in an emergency. In an emergency, blood grouping can be done easily and quickly in 2 or 3 minutes in the laboratory on glass slides with appropriate reagents, by trained technical staff. This method depends on the presence or absence of agglutination, which can usually be visualized directly, although occasionally a light microscope may be needed. If laboratory services are not available, another system of deciding which type of blood to use in an emergency is the bedside card method of blood grouping, where a drop of the intended recipients' blood is added to dried reagents on a prepared card. This method may not be as reliable as laboratory methods, which are preferable.

Disclaimers CROSSMATCH

General Information

The Crossmatch also known as compatibility testing, pretransfusion testing or type and crossmatch (Type and Cross; T & C). The definition of a compatibility test (crossmatch) is a series of procedures use to give an indication of blood group compatibility between the donor and the recipient and to detect irregular antibodies in the recipient's serum.

The main purpose for performing a crossmatch is to promote (not ensure) the safe transfusion of blood. We are performing testing to the best of our ability that will demonstrate that the donor blood is compatible with the recipient's blood. Crossmatch procedures should be designed for speed and accuracy - get the safest blood reasonably possible available to the patient as soon as possible. In summary, the AABB Technical Manual states the goals of a compatibility test is to:

Detect as many clinically significant antibodies as possible

Detect as few clinically insignificant antibodies as possible

Complete the procedure in a timely manner. (p. 379)

Once donor blood is crossmatched with a potential recipient, the results of the crossmatch is good only 3 days. If the physician wants the donor blood available longer, we must get a new recipient sample and repeat tests. This protocol helps detect new antibodies that may be forming, especially when patient has been transfused within past three months.

Parts of the Crossmatch

The AABB Standards for Blood Banks and Transfusion Services requires that certain procedures are performed before blood is transfused to a recipient:

Identification of the recipient and recipient blood sample is crucial since the major of the hemolytic transfusion reactions are due to errors in patient or sample identification.

ABO and Rh typing of the recipient's blood and resolving any ABO discrepancies. If the discrepancy can not be resolved before the patient needs the transfusion type O blood should be given. If problems arise with the D testing, Rh negative blood should be given.

Performing an antibody screen on the recipient's serum for clinically significant antibodies. These antibodies are most likely to occur in the 37oC and AGT phases of testing. Each negative AGT test must be followed by "Coombs Control Check Cells." An autocontrol may or may not be used. Some labs prefer to perform this routinely during the antibody screen while others will only include it if an antibody needs to be identified. The autocontrol has to be part of the antibody identification procedure. The SOP of each institution must be followed by all individuals performing these tests.

Comparing present findings with previous records for the recipient. If previous testing has been performed on the recipient and should match current testing. These comparisons can give assurance that no identification errors have occurred, but it is not proof. Records would also show if clinically significant antibodies have been detected in the past. These antibodies may be presently at

undetectable levels. Any history of clinically significant antibodies, even if undetectable now in the patient, dictates an antiglobulin phase crossmatch needs to be done between the recipient's serum and the donor's cells.

Confirmation of the ABO and Rh type of the red cell components being given when the shipment of blood is received in the laboratory.

Selection of appropriate ABO and Rh component units for the recipient first would be the same ABO and Rh type. Transfused donor red cells must be ABO compatible with the patient's plasma and whatever antibodies may be present. Transfused plasma must be ABO compatible with the recipient's red cells. AABB Technical Manual's Table 18-2

Selection of Components When ABO-Identical Donors Are Not Available, p 385

ABO Requirements

Whole BloodMust be identical to that of the recipient

Red Blood Cells (most plasma removed) Must be compatible with the recipient's plasma.

Granulocytes, Pheresis Must be compatible with the recipient's plasma.

Fresh Frozen Plasma Must be compatible with the patient's red cells.

Platelets, Pheresis All blood groups acceptable; components compatible with the recipient's red cells preferred

Cryoprecipitated AHFAll ABO groups acceptable

Rh-positive components should be given to Rh-positive individuals and Rh-negative units should be reserved for D-negative individuals. The physician needs to be involved in any decisions relating to giving Rh-positive blood to an Rh-negative individuals since those individuals have an 80% chance of making an anti-D following transfusion.

Perform a crossmatch either serologically or via a computer. If no clinically significant antibodies are found in the recipient the institution has the option of choosing an immediate-spin crossmatch (serologic technique) or a

computer crossmatch. If clinically significant antibodies are found, an antiglobulin crossmatch must be performed.

Label the components with the recipient's identifying information

Type and Screen

The type and screen consists of ABO/Rh, antibody screen, and a records check. This order is used when likelihood of needing blood is low. Therefore, no donor blood crossmatched to patient. If need for blood suddenly arises, you can take sample that is already typed and screened, and perform a crossmatch with donor units from the specimen. Type and screen protocol cannot be used if patient has an antibody. Then an antiglobulin crossmatch must be performed.

Benefits of a Crossmatch

Performing a crossmatch before transfusing blood has the following benefits:

Detects major ABO errors (ie. crossmatching an A donor with an O or B recipient )

Detects most recipient antibodies to antigens on donor red cells (if the antibody is in high enough titer to react) One of the most common clinically significant antibodies that are missed are the Kidd antibodies.

Limitations of a Crossmatch:

A crossmatch also has limitations:

Will not detect errors in patient identification (unless a previous record exists)

Will not detect ABO mix-ups if blood types are compatible (can crossmatch group A donor blood for an AB recipient)

Will not detect Rh errors (can crossmatch Rh+ donor blood with Rh negative recipient with no reaction if the patient has no anti-D)

Will not detect all recipient antibodies to donor antigens (antibody may be too weak to detect, but still cause transfusion reaction such as the Kidd antibodies)

Will not prevent alloimmunization of recipient (only ABO and Rh antigens matched - patient can potentially

make antibody to all the other antigens) This is why many of the discovered antibodies are found in multi-transfused patients.

Immediate Spin versus Antiglobulin, Coombs, Crossmatch

The purpose of Immediate spin step of crossmatch is to detect major ABO incompatibility between donor and recipient. ABO incompatibility is the most common life-threatening type of transfusion reaction and is often due to clerical errors.

It is permissible to stop at immediate spin step of crossmatch if:

Immediate spin is negative and

Antibody screen is negative in all phases and

There is no record of previous antibodies

It is NOT permissible to stop at the immediate spin step and you must incubate and carry crossmatch through antiglobulin, Coombs, phase if:

Immediate Spin test agglutinated or

Patient has an antibody (screening cells are positive) or

Patient has a record of a previous antibody

Benefits of an Immediate Spin only crossmatch:

Makes blood available to patient faster

More cost-effective

90% of patients are eligible for immediate-spin crossmatches

Electronic or Computer Crossmatch As An Alternative to the immediate-spin crossmatch

An institution may choose to perform computer crossmatches instead of an immediate-spin crossmatch. They must meet specific criteria in order to do electronic

crossmatches. Electronic crossmatches have no mixing of patient serum and donor cells in test tube - computer verifies ABO/Rh compatibility of donor and recipient

Computer system must be FDA-approved and validated to do this

Patient ABO/Rh must have been typed at least twice - by two different technologists

Patient has no antibodies and no record of previous antibodies

Donor information must be bar-coded into computer inventory for accuracy

Computer does not allow use of donor unit until its ABO/Rh is verified

Computer does not allow issue of ABO/Rh incompatible blood

Crossmatch Problems:

OBJECTIVES - COMPATIBILITY TESTING

Discuss the steps in compatibility testing and explain their purpose.

Discuss the reasons for compatibility testing.

Discuss the limitations of compatibility testing.

Explain what a Type and Screen consists of, and when it would be used.

Explain when a LISS-Coombs crossmatch would be done versus an immediate-spin crossmatch.

Explain what an electronic crossmatch is.

Describe what additional testing must be done when crossmatching a patient with an antibody.

Describe how to determine the number of units to screen when crossmatching blood for a patient with an antibody

Discuss how to resolve the following problems encountered in compatibility testing:

Test results not matching previous records

Screening cells positive at room temperature, negative in Coombs

Screening cells positive in Coombs only - new antibody

Screening cells positive in Coombs only - previously identified antibody

Screening cells positive both at RT and in Coombs

Negative screening cells, but records show a previously-identified antibody

Negative screening cells but crossmatch positive at immediate spin

Positve autocontrol

State how long you may keep blood crossmatched, before having to get a new sample and repeat the test

Chart Of A-B-O Blood Donor & Recipient Compatibility

RECIPIENT

D

O

N

O

R Alleles &

Antibodies O

anti-A

anti-B A

anti-B B

anti-A AB

None

O None None None None

A Clump None Clump None

B Clump Clump None None

AB Clump Clump Clump None

Chart of A-B-O Blood Donor-Recipient Compatibility. Serious problems may arise when the antibodies of the recipient clump the blood cells of the donor. [The reverse scenario is not as serious because the antibodies of the donor are diluted by the recipient's blood volume.] Clumping of the donor's blood is indicated by the word "Clump" in the red squares. No clumping of the donor's blood is indicated by the word "None" in the green squares. None also denotes the lack of anti-A or anti-B antibodies in the type O recipient. It is clear from this chart that the "universal donor" is type O, while the "universal recipient" is type AB. If you include the Rh factor, then the universal donor becomes O Negative while the universal recipient becomes AB Positive.

Simplified Explanation For Rh Blood Factor

Rh Neg

Blood

Add a drop of anti-Rh antibodies (anti-D serum) to a drop of blood

on a slide labeled Rh. The Rh negative blood (shown above) will

not clump, while the Rh positive blood (below) will show clumping:

Rh Pos

Blood

Placing the Rh slide on a warming box will hasten the agglutination reaction. Backlighting will also make it easier to see the clusters of agglutinated red blood cells that appear like minute grains of sand in the blood. Rocking the slide back and forth also makes it easier to see the grainy texture of the agglutinated blood.

An Rh Blood Typing (Warming) Box.

Remember that the anti-Rh serum will only agglutinate the positive D factor. There are technically three positive genes called C, D and E. The negative alleles for these three genes are usually denoted by small case c, d and e. This is an example of multiple gene (polygenic) inheritance which is explained in more detail at the following URL:

Multiple Gene Inheritance In The Rh Factor

Although it is much more complicated, the Rh blood factor can be explained by a pair of alleles on homologous chromosome pair #1. The dominant Rh positive gene (+) produces the Rh antigen, a glycoprotein constituent of the RBC membrane (see above Rh positive RBC illustration). Like the type O

gene, the recessive Rh negative gene (-) does not produce an antigen. The following table summarizes Rh inheritance in humans:

Blood Phenotype Blood Genotype Antigen on

RBC Membrane Immune (IgG)

Antibodies

Rh Positive

(85% of U.S.) + + or + - Rh antigen None

Rh Negative

(15% of U.S.) - - No antigen May Produce

anti-Rh

If Rh positive blood is accidentally given to an Rh negative recipient, the recipient will begin producing anti-Rh antibodies. Because of the time factor involved in building up a concentration (titre) of antibodies, the first transfusion may not cause any major problems; however, a subsequent transfusion of Rh positive blood could be very serious because the recipient will clump all of the incoming blood cells. The donor-recipient scenario with Rh blood types is summarized in the following table:

Donor Recipient Anti-Rh Antibodies

in Recipient's Blood

Rh Positive Rh Negative Will Produce

anti-Rh Antibodies

Rh Negative Rh Positive Will Not Produce

anti-Rh Antibodies

Since Rh negative people may produce anti-Rh antibodies, Rh positive blood should not be given to an Rh negative recipient. Based upon the above table, Rh positive recipients can theoretically receive positive or negative blood, and Rh negative donors can theoretically give to Rh positive and Rh negative recipients. Therefore, the "universal donor" is O Negative, while the "universal recipient" is AB Positive.

Anti-Rh (immune-type) antibodies can readily pass through the placental capillary membranes. A serious potential problem called maternal-fetal blood incompatibility or Rh Disease could occur with a pregnant Rh negative mother who carries an Rh positive fetus. Leakage of fetal red blood cells (RBCs) into the mother's system through minute lesions in the placenta may cause her to produce anti-Rh antibodies. This could occur during the latter months of pregnancy or when the baby is delivered. Because of the time interval involved in producing a concentration (titre) of antibodies, the first Rh positive child may not be adversely affected. However, a subsequent Rh positive child may be at risk because the mother's anti-Rh can pass through the placenta, thus entering the fetal circulatory system and clumping fetal RBCs.

The medical term for this maternal-fetal condition is "erythroblastosis fetalis" because of the presence of nucleated, immature RBCs called erythrobasts in the fetal circulatory system. The fetus bone marrow releases immature erythroblasts because of the destruction of mature RBCs (erythrocytes) by the mother's anti-Rh antibodies. RhoGam®, a serum containing anti-Rh antibodies, is now given to Rh negative woman within 72 hours after giving birth to their Rh positive baby. The RhoGam® enters the mother's circulatory system and destroys any residual fetal positive RBCs that may be present in her system. This prevents her from producing anti-Rh antibodies. RhoGam® must be given after each Rh positive baby. In this scenario of erythroblastosis fetalis, the fetus must be Rh positive, the mother Rh negative and the father Rh positive. You can easily determine the exact genotype of the mother and fetus, but the father's genotype could be homozygous or heterozygous Rh positive. Rh incompatibility is summarized in the following table:

Rh Pos Father

+ + or + - X Rh Neg Mother

- -

1st Rh Pos Child

+ -

Rh positive RBCs from the fetus enter the mother's circulatory system.

After several days, the mother begins to produce anti-Rh antibodies.

2nd Rh Pos Child

+ -

Anti-Rh antibodies from mother pass through placenta and enter fetal

circulatory system. The antibodies begin clumping fetal positive RBCs.

There are also reported cases of maternal-fetal blood incompatibility with the A-B-O blood groups; however, the Rh factor appears to be much more common. The larger anti-A and anti-B antibodies (IgM type) with molecular weights of 950,000, apparently don't penetrate the placental membranes as readily. In the case of A-B-O blood incompatibility, the anti-A and anti-B antibodies of a type O mother may enter the circulatory system of a Type A or Type B fetus, thus causing agglutination of the fetal RBCs. If the fetal blood cells just happened to be Rh positive and entered the mother's circulatory system, they would be destroyed by the mother's anti-A or anti-B antibodies before her system began to produce anti-Rh immune-type (IgG) antibodies. In this latter case, the anti-A or anti-B antibodies would actually serve as a natural immunity to Rh maternal-fetal blood incompatibility.

Like most topics in biology, the true life explanation is a lot more complicated. Rh inheritance is no exception. It actually involves three different pairs of genes at three different loci on homologous chromosome pair #1. The gene pairs are C & c, D & d and E & e. The terms "positive" and "negative" essentially refer to the D factor, so homozygous DD and heterozygous Dd are positive, while homozygous recessive dd is negative. For a more in depth explanation of this interesting example of polygenic inheritance, please refer to the following hyperlink:

Rh Factor: Another Example Of Polygenic Inheritance

Rh Factor: Another interesting example of polygenic inheritance is the Rh factor. Unlike the A-B-O blood types where all the alleles occur on one pair of loci on chromosome pair #9, the Rh factor involves three different pairs of alleles located on three different loci on chromosome pair #1. In the following diagram, 3 pairs of Rh alleles (C & c, D & d, E & e) occur at 3 different loci on homologous chromosome pair #1. Possible genotypes will have one C or c, one D or d, and one E or e from each chromosome. For example: CDE/cde; CdE/cDe; cde/cde; CDe/CdE; etc.

In order to determine how many different genotypes are possible, you must first determine how many different gametes are possible for each parent, then match all the gametes in a genetic checkerboard (See the following Table 3). Although the three pairs of genes are linked to one homologous pair of chromosomes, there are a total of eight different possible gametes for each parent: CDE, CDe, CdE, Cde, cDE, cDe, cdE, and cde. This number of gametes is based on all the total possible ways these genes can be inherited on each chromosome of homologous pair #1. [It is not based on the random assortment of these genes during meiosis in the parents because all three genes are closely linked together on the same chromosome; therefore, all three genes tend to appear together in the same two gametes: CDE and cde.] The possible different genotypes are shown in the following Table 3:

Gametes CDE CDe CdE Cde cDE cDe cdE cde

CDE CDE/

CDE CDE/

CDe CDE/

CdE CDE/

Cde CDE/

cDE CDE/

cDe CDE/

cdE CDE/

cde

CDe CDe/

CDE CDe/

CDe CDe/

CdE CDe/

Cde CDe/

cDE CDe/

cDe CDe/

cdE CDe/

cde

CdE CdE/

CDE CdE/

CDe CdE/

CdE CdE/

Cde CdE/

cDE CdE/

cDe CdE/

cdE CdE/

cde

Cde Cde/

CDE Cde/

CDe Cde/

CdE Cde/

Cde Cde/

cDE Cde/

cDe Cde/

cdE Cde/

cde

cDE cDE/

CDE cDE/

CDe cDE/

CdE cDE/

Cde cDE/

cDE cDE/

cDe cDE/

cdE cDE/

cde

cDe cDe/

CDE cDe/

CDe cDe/

CdE cDe/

Cde cDe/

cDE cDe/

cDe cDe/

cdE cDe/

cde

cdE cdE/

CDE cdE/

CDe cdE/

CdE cdE/

Cde cdE/

cDE cdE/

cDe cdE/

cdE cdE/

cde

cde cde/

CDE cde/

CDe cde/

CdE cde/

Cde cde/

cDE cde/

cDe cde/

cdE cde/

cde

Polygenic inheritance in the Rh blood factor. Every genotypic combination with DD or Dd is classified as Rh Positive (red). This is about 85% of the U.S. population because the D gene is more common than the C and E genes. Every genotypic combination with dd is classified as Rh Negative (blue). Since the ratio of C and E genes is much less than D genes, approximately 15% of the U.S. population are Rh negative (dd). Consolidating the duplicates, a total of 10 genotypes are homozygous recessive for the d allele (dd); however, nine of these genotypes are actually positive for the C and E factors: Cde/cde (0.46%), Cde/Cde (0.0036%), cdE/cde (0.38%), cdE/cdE (0.0025%), Cde/cdE (0.006%), CdE/cde (0.008%), CdE/Cde (0.0001%), CdE/cdE (0.0001%), and CdE/CdE (0.00001%). Therefore, only about 0.86% of the U.S. population are positive for C and E. Expressed as a decimal, this is 0.0086 or 8.6 out of 1000. This is why Rh incompatibility involving the C and E genes is rare in the U.S. population.

Other Examples Of Polygenic Inheritance

Antigen Immune Antibodies (In Blood Plasma)

(RBC Membrane) anti-C anti--D anti-E

C (RhC) ------ ------ ------

D (RhD) ------ RhoGAM & Biology TypingSerum ------

E (RhE) ------ ------ ------

Rh antibodies primarily utilized in immunoglobulin serums.

More than 98% of all cases of hemolytic disease of the newborn (maternal-fetal blood incompatibility) are caused by the D antigen, also referred to as RhD and Rh Positive (+). This is why RhoGam and standard blood typing kits for general biology labs only contain anti-RhD (anti-D) antibodies. Anti-C and anti-E antibodies against the C and E antigens can be associated with maternal-fetal blood incompatibility, but this is uncommon and only occurs in a small percentage of non-RhD cases.

Apparently immune globulins (such as RhoGam) are not available to prevent these rare cases. According to Dr. Kenneth J. Moise, Jr., Director of the Division of Maternal-Fetal Medicine at University of North Carolina Medical School at Chapel Hill, more than 43 other RBC antigens have been implicated in the non RhD cases. Especially problematic are the Kell (K1), c, Duffy (Fya) and Kidd (Jka and Jkb) antigens. A recent study from a tertiary referral center in New York found 550 cases of antibodies associated with hemolytic disease of the newborn in 37,506 blood samples taken from women of reproductive age (1.1% incidence). Anti-D occurred in 25% of the samples, anti-Kell in 28%, anti-c in 7%, anti-Duffy in 7%, anti-Kidd in 2%, anti-E in 18%, anti-C in 6%, anti-MNS in 6%, and anti-Lutheran in 2%. The following link contains a summary of Rh maternal-fetal blood incompatibility from the UNC Department of Obstetrics and Gynecology:

Information Abou

University of IowaHospitals and Clinics

COURSES/HOMEPLANNINGNEWSLETTERS

00979071757102 FORID:11 Search

www .medicine.u

ABOUTFAQ

Hematology in the Physician Office Laboratory Section I

Practice Test (No Cost) | CME Credit Test (20 module Course Registration = 425.00)

Kathleen Kelly B.S., MT(ASCP)

ObjectivesSection I

I. IntroductionII. SamplesIII. Complete Blood Count (CBC)

A. Table Top Analyzers

1. Cell Counts2. Hemoglobin3. Hematocrit4. Correlating Hemoglobin and Hematocrit Values5. Comparing Automated and Manual Hematocrits

B. Manual Methods

1. Cell counts2. Hematocrits

a. quality assurance

3. Hemoglobins

C. Red Cell and Platelet Indices

Objectives

Kathleen Kelly B.S., MT(ASCP)

Upon completion of this module, the participant will be able to:

1. State the preferred anticoagulant for most hematologic testing.

2. Discuss the most common principles utilized in table top hematology analyzers.

a. Cell counts

b. Hemoglobin

c. Hematocrit

3. Briefly discuss sources of error in CBC results.

a. Cell counts

b. Hemoglobin

c. Hematocrit

4. Correlate hemoglobin and hematocrit results.

5. Discuss the use and reliability of hemacytometer cell counts, centrifuged hematocrits, and alternative

hemoglobin procedures.

6. Discuss the centrifuged hematocrit, including sources of error, preventive maintenance, and quality control.

7. Discuss the differences between automated and centrifuged hematocrits.

8. Discuss the derivation and use of red cell indices.

Section Top | Title Page

I.Introduction

Kathleen Kelly B.S., MT(ASCP)

Hematology testing has long been part of the Physician office laboratory testing. Historically, the centrifuged

hematocrit has been one of the most popular laboratory tests. The value of this test has not diminished; however

the availability of cost-effective tabletop instrumentation has put the manual hematocrit in the back seat.

Simple equipment that requires minimal operator intervention has made a complete blood count (CBC) readily

available. The through put of these instruments is fast enough to give cell counts, indices, hemoglobin, hematocrit,

and even a differential in the same time it takes to perform a manual hematocrit. Most of the instruments

currently marketed to physician office laboratories are specifically designed to be user friendly and can be

purchased with ready to use charts and procedures for the required preventive maintenance and quality

assurance.

This unit will discuss the hematology testing most often done in a physician office laboratory. The intent is to

discuss general information on instrumentation and commonly performed tests. Specific manufacturer's

information will not be included. This can be obtained from the individual manufacturer.

Section Top | Title Page

II. Samples

Kathleen Kelly B.S., MT(ASCP)

Sample procurement is addressed in the phlebotomy module, but proper sample handling can not be over

emphasized. Most hematology testing is done on blood anticoagulated with di- or tri- potassium EDTA (ethylene

diamine triacetic acid). Laboratories in the U.S.A. have commonly used K3EDTA in liquid form in a glass or plastic

tube. The International Council for Standards in Hematology recommends K2EDTA sprayed on the walls of plastic

collection tubes.1 The decision to change anticoagulants requires consideration of manufacturer's directions,

reference laboratory preferences, reference ranges, and available literature.

Bullet tubes, with EDTA, are available for collecting blood from skin punctures. The bullet tube must fill quickly and

the blood mixed with the EDTA before coagulation begins. The quality of the skin puncture sample is very

technique dependent.

Samples collected in EDTA must be well mixed at the time of draw and before sampling. The correct mixing time

before sampling is five minutes on an aliquot mixer or, if they are mixed manually, a minimum of sixty inversions.

However, avoid over mixing. Prolonged mixing on aliquot mixers causes hemolysis.

As a safety precaution, always open evacuated tubes under a protective shield. Residual vacuum causes

aerosols.

1. Recommendations of the ICSH for EDTA anticoagulation of blood for blood cell counting and sizing. Amer J Clin

Pathol. 1993:100:371

Brunson D, Smith D, Bak A, et al. Comparing hematology aK2EDTA vs. K3EDT. Laboratory Hematology 1995, 1:112-

119.

Section Top | Title Page

Protective Shields

Kathleen Kelly B.S., MT(ASCP)

These are examples of freestanding protective shields. They are available through laboratory supply and safety

product distributors. Previous Page |

Section Top |

Title Page

III. Complete Blood Count (CBC)

Kathleen Kelly B.S., MT(ASCP)

The CBC is the meat and potatoes of hematology. Over the years, the components of the CBC have expanded, as

the instrumentation has become more sophisticated. The CBC test menu on basic instruments usually includes cell

counts (red cells, white cells, and platelets), hemoglobin, hematocrit, and red cell indices. More complex

instrumentation adds automated differentials, platelet indices, and white cell differentials. Performing complete

blood counts using multiple manual or semi-automated methods is no longer cost or time effective. The manual

and semi-automated methods are useful for measuring single parameters and as back up procedures.

A. Table Top Analyzers

These analyzers vary in the extent of the automation involved. The simplest analyzers require the operator to mix

the sample and present an open sample tube to the instrument. Sampling units that pierce the cap of the tube

save time, are readily available, and are safer. The more sophisticated systems read bar coded labels, mix, and

sample through the cap of the tube. Skin puncture samples may need to be diluted and/or presented to a different

sampling probe. The ease of testing skin puncture samples and 2 or 3 ml tubes may be an important consideration

in choosing an instrument. The top of the line hematology systems can make peripheral smears, store thousands

of results, and store and manipulate quality control data. Most of the instruments can be interfaced with a

laboratory information system.

The following URLs will connect to some of the manufacturers of table top hematology analyzers.

1. http://www.beckmancoulter.com/products/instrument/hematology/actseries.asp

Beckman Coulter

2. http://www.bayerdiag.com/

Bayer Diagnostics

3. http://www.sysmex.com/usa/ourbusiness/ourbusiness.cfm?dis_id=1

Dade Sysmex

Section Top | Title Page

1. Cell Counts

Kathleen Kelly B.S., MT(ASCP)

On any given instrument, all of the cell counts are based on the same principle. The Coulter (principle of electrical

impedance or a modification is the most common principle used in the smaller hematology instruments. This

principle is based on the ability of the saline diluent to conduct electricity while the suspended cells are

nonconductive. As the cells pass through an aperture they break the current between the external and internal

electrode and are enumerated and sized. Red cells and white cells are counted in separate baths or channels, with

the red cells lysed when white cells are counted. Platelets are usually counted with the red cells and the cells are

differentiated by size. All cells are reported in units per volume of whole blood.

Red cell histograms derived from these counts have a small tail to the right of the curve. This

represents coincidence, which is multiple cells passing through the aperture at the same time. The correction for

coincidence takes place in the cell count calculation and is not a concern.

The other basic counting principle is based on light scatter. Here, a single cell passes through a beam of light from a

laser or tungsten-halogen light source. The cell is counted as it breaks the beam of light and the light is scattered.

The scattered light is measured and translated into cell size. The counts are reported in units per volume of whole

blood.

Some hematology instrumentation prints the red cell and platelet histograms as part of the report. These

histograms give a visual interpretation of the cell population and correlate with the indices.

A third type of instrument is advertised as a dry system that works by centrifugation. It generates the cell counts,

hemoglobin, and hematocrit from a centrifuged specimen. These are light, portable instruments made by Becton

Dickinson. The rest of this discussion does not apply to this type of instrument.

Sources of error in cell counts include:

1. Cold agglutinins - low red cell counts and high MCVs can be caused by a decreased number of large red cells or

red cell agglutinates. If agglutinated red cells are present, the automated hematocrits and MCHCs are also

incorrect. Cold agglutinins cause agglutination of the red cells as the blood cools. Cold agglutinins can be present in

a number of disease states, including infectious mononucleosis and mycoplasma pneumonia infections. If red cell

agglutinates are seen on the peripheral smear, warm the sample in a 37 degrees C heating block and mix and test

the sample while it is warm. Strong cold agglutinins may not disperse and need to be redrawn in a pre-warmed

tube and kept at body temperature.

2. Fragmented or very microcytic red cells these may cause red cell counts to be decreased and may flag

the platelet count as the red cells become closer in size to the platelets and cause an abnormal platelet histogram.

The population is visible at the left side of the red cell histogram and the right end of the platelet histogram.

3. Platelet clumps and platelet satellitosis - these cause falsely decreased platelet counts.

Platelet clumps can be seen on the right side of the platelet histogram. Decreased platelet counts are confirmed by

reviewing the peripheral smear. Always scan the edge of the smear when checking low platelet counts.

4. Giant platelets - these are platelets that approach or exceed the size of the red cells. They cause the

right hand tail of the histogram to remain elevated and may be seen at the left of the red cell histogram.

5. Nucleated red blood cells - these interfere with the WBC on some instruments by being counted as

white cells/lymphocytes

Red cell histograms - histograms are derived by plotting the size of each red cell on x axis and the relative number

on the y axis. They are used to determine the average size, distribution of size, and to detect subpopulations.

Section Top | Title Page

Red Cell Histogram

Kathleen Kelly B.S., MT(ASCP)

Red cell histograms are derived by plotting the size of each red cell on x axis and the relative number on the y axis.

They are used to determine the average size, distribution of size, and to detect subpopulations. This histogram

represents a normal red cell distribution. The small tail to the right of the curve represents coincidence, multiple

cells passing through the aperture at the same time. Previous Page |

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Agglutinated Red Blood Cells

Kathleen Kelly B.S., MT(ASCP)

The arrow points to a red cell agglutinate. Previous Page |

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Microcytic and Fragmented Red Cells

Kathleen Kelly B.S., MT(ASCP)

Previous Page |

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Red Cell and Platelet Histograms

Kathleen Kelly B.S., MT(ASCP)

The black curves on the red cell and platelet histograms indicate ìexpectedî or normal cell distributions. The red

curves demonstrate the effect of very microcytic red cells on the histograms. Since microcytic red cells only effect

the right end of the platelet curve, the black and red lines are superimposed for most of the curve. Previous Page |

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Platelet clumps

Kathleen Kelly B.S., MT(ASCP)

There is a large clump of platelets in the center of the field. Previous Page |

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Platelet Satellitosis

Kathleen Kelly B.S., MT(ASCP)

The neutrophils in the center of the field are surrounded by platelets. This is an uncommon phenomenon seen in

EDTA samples. The platelets surround the neutrophils and "stick". This results in a false decrease in the platelet

count. Previous Page |

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Giant Platelets

Kathleen Kelly B.S., MT(ASCP)

Platelets that are 6 microns in diameter or larger, are considered giant platelets. Multiple giant platelets are visible

in this field. Previous Page |

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Red Cell and Platelet Histograms

Kathleen Kelly B.S., MT(ASCP)

The black curves on the red cell and platelet histograms indicate "expected" or normal cell distributions. The red

curves demonstrate the effect of giant platelets on the red cell and platelet histograms. Previous Page |

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Nucleated Red Blood Cells

Kathleen Kelly B.S., MT(ASCP)

The two nucleated cells are immature red cells that have been released from the bone marrow ahead of schedule.

Previous Page |

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2. Hemoglobin

Kathleen Kelly B.S., MT(ASCP)

Hemoglobin, on most automated systems, is measured as cyanmethemoglobin. This is a long-standing method

that reliably measures all the hemoglobin variants except sulfhemoglobin. However, some manufacturers indicate

that their instruments now include sulfhemoglobin in the total hemoglobin measurement.

Red cells are lysed and potassium ferricyanide oxidizes hemoglobin to methemoglobin, which combines with

potassium cyanide forming cyanmethemoglobin. The brown color is measured spectrophotometrically and the

corresponding hemoglobin reported. The end point of the reaction is stable and the reaction is linear to 20 g/dL or

higher. Reagents for cyanmethemoglobin are light sensitive and poisonous.

However, the Sysmex systems can use a sulfhemoglobin methodology to measure total hemoglobin. The reagent

sodium lauryl sulfate disrupts the red cell membrane and binds to the globin chains, causing methemoglobin to be

formed. This binds to the sulfate group producing a conjugate with an absorption peak at 535 nm. No hazardous

waste is produced.

Common sources of error in measuring hemoglobin include anything that will cause turbidity and interfere with a

spectophotometric method. Examples are a very high WBC or platelet count, lipemia and hemoglobins that are

resistant to lysis, such as hemoglobins S and C. The hemoglobin will be falsely increased with turbidity.

Manufacturer's guidelines address interferences.

A simple method for obtaining a hemoglobin value from a lipemic sample is to use plasma replacement.

Plasma replacement - centrifuge a whole blood specimen and remove the plasma with an automatic pipettor.

Record the volume removed and, with the same pipettor and a clean tip, replace the exact volume removed with

isotonic saline. Mix the sample well and retest.

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3. Hematocrit

Kathleen Kelly B.S., MT(ASCP)

A hematocrit is the volume of the red cells as compared to the volume of the whole blood sample. Hematocrits on

the automated systems are calculated. The volume of each red cell is measured as it is counted and a mean cell

volume is derived. The calculations are not precisely the same. But, they can be summarized as mean corpuscular

red cell volume (MCV) multiplied by the red cell count (RBC). Hematocrits are reported in L/L or the traditional %.

When electrolyte displacement is used, the resulting hematocrit is sometimes called the CCV, or conductance cell

volume.

Hematocrits calculated by automated instruments depend on correct red cell counts and red cell volumes to arrive

at an accurate hematocrit. Hence, anything affecting the red cell count or volume measurement will affect the

hematocrit. This method is not as sensitive to the ratio of blood to EDTA as the centrifuged hematocrit. Since the

red cells are resuspended in isotonic saline, they regain their normal shape and size. The practical side of this is

that a reliable automated hematocrit can frequently be obtained from a short sample.

It is very useful to have a hematocrit centrifuge for back up. It can be used when the main instrument is down and

to trouble shoot or check hemoglobins that do not correlate with the other results.

short sample - an evacuated tube that is not full or does not have enough blood to satisfy the manufacturer's

recommended anticoagulant to blood ratio.

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4. Correlating Hemoglobin and Hematocrit Values

Kathleen Kelly B.S., MT(ASCP)

The hemoglobin times three roughly equals the hematocrit in most patients.

Example:

14.8 x 3 = 44 (patient's hematocrit result is 45 L/L)

11.0 x 3 = 33 (patient's hematocrit result is 32 L/L)

The exception to this rule is in patients with hypochromic red cells. These patients will have

hematocrits that are more than three times the hemoglobin.

Hypochromic red cells- red cells that have an increased zone of pallor on Wright's stain. These cells contain

decreased hemoglobin for their size and have a decreased MCHC.

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Hypochromic Red Cells

Kathleen Kelly B.S., MT(ASCP)

Hypochromic red cells have an increased zone of pallor on Wright's stain. These cells contain decreased

hemoglobin for their size and have a decreased MCHC. Previous Page |

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5. Comparing Manual and Automated Hematocrits

Kathleen Kelly B.S., MT(ASCP)

Understanding the differences in hematocrit methodology helps explain why some patient's hematocrits vary

more than others do when comparing an automated hematocrit with a centrifuged hematocrit. Theoretically,

measuring each red cell and adding those volumes to get a total red cell volume is the most accurate method.

However, we use the centrifuged hematocrit as our "gold standard" and may even calibrate the automated

hematocrit to match the packed cell volume (PCV). Centrifuged hematocrits have a built in bias due to the trapped

plasma. When red cells have very abnormal shapes, this trapped plasma may be increased enough to cause a

significant change in the centrifuged hematocrit. Some studies have shown that the two methods show better

correlation if K2EDTA is used as the anticoagulant.

Understanding how the automated hematocrit is derived is very important when trouble shooting or using back up

methods such as a centrifuged hematocrit. If the hematocrits appear incorrect on the automated equipment, it

means that the MCV and/or red cell count are also suspect. If a centrifuged hematocrit is substituted for an

automated value, the MCV must be manually calculated.

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1. Cell Counts

Kathleen Kelly B.S., MT(ASCP)

The UNOPETTE® (system has made manual cell counts much easier and more reliable, but hemacytometer cell

counts are still very technique dependent. Manual cell counts should always be diluted and counted in duplicate.

The two WBC dilutions must match within 10% and platelet dilutions must match within 20%. Manual platelet

counts are more difficult than manual white cell counts, especially without the aid of a phase microscope.

Errors occur in all three steps of the procedure; diluting, plating, and counting. Dilution errors occur when the

pipettes are under-filled, not wiped correctly, or blood is left in the neck of the reservoir. Plating errors include

errors in expelling well-mixed sample, filling technique, and handling the hemacytometer between filling and

counting. Scanning each chamber for even cell distribution before it is counted can help identify some errors.

Counting errors include choosing an inappropriate area to count based on the number of cells present, improper

counting technique, missing cells, counting junk, and misidentifying cells. Manual WBC and platelet counts should

be confirmed with an estimate performed on a Wright's stained peripheral smear.

Manual red counts are inaccurate and imprecise. The hemoglobin and/or hematocrit are usually adequate if a CBC

is not available.

Using manual cell counts for back up is usually more trouble than it is worth. Besides requiring excellent technique,

UNOPETTES® outdate and are expensive. The procedure is time consuming. In a pinch it may be preferable to look

at a well prepared, stained peripheral smear. An increase in white cells, the presence of immature cells, or reactive

lymphocytes may answer the immediate question.

Quality control of manual cell counts is time consuming. At least two levels of controls for all cell types counted

must be counted on every shift and the diluent from the vials must be examined in the hemacytometer to ensure

that there is nothing contaminating the diluent that might be counted as a cell. It is also necessary to do counts on

proficiency samples and to ensure that everyone who is performing counts is getting equivalent results, i.e.,

competency testing.

Hemacytometers are necessary for body fluid counts. Laboratories that examine synovial and other body fluids

may perform cell counts as well as a crystal analysis performed on a polarized microscope.

In summary, if you do not do manual cell counts on blood samples, do not start.

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2. Hematocrits

Kathleen Kelly B.S., MT(ASCP)

When considering back up equipment, the hematocrit centrifuge is probably the most useful. It requires little

expenditure after the initial cost and has simple preventive maintenance and quality control (PM and QC)

requirements.

A centrifuged hematocrit is also called a packed cell volume (PCV). As in the automated hematocrit, a hematocrit is

the volume of the red cells as compared to the volume of the whole blood sample and is reported in L/L or as a %.

The packed cell volume is determined by centrifuging the specimen in capillary tubes and measuring the height of

the red cell column.

The sample is venous blood drawn in EDTA or capillary blood collected in heparinized (red banded)

microhematocrit tubes. A short EDTA sample will have an increased anticoagulant to red cell ratio, which causes

the red cells to shrink and the hematocrit to be falsely decreased. EDTA must not exceed 2 mg/ml of whole blood.

For EDTA samples use blue banded, plain capillary tubes. Traditional tubes are glass and unplugged. Mylar coated

tubes, plastic tubes, and pre-plugged tubes are available. Plastic pre-plugged tubes are a good choice. They

decrease the risk of breaking a tube and puncuturing the finger of the person performing the test and avoid some

sources of error.

To perform a microhematocrit:

1. Fill two red or blue banded capillary tubes, from the end without the colored band, 1/2 to 2/3 full. Tilt each tube

so that the blood is near the colored band. Hold the tube horizontally and wipe all of the excess blood off of the

tubes before it dries. Be careful not to wipe across the end of the tube. Absorbent material will pull out more

plasma than cells.

2. For unplugged tubes, hold each tube horizontally and seal the end with the colored band by inserting it into the

clay. This is the fire-polished end. Add the sealant until it is just above or below the colored band.

Filling and sealing this way keeps the clay from becoming contaminated with blood, helps prevent leakage, is safer

for the person testing, and keeps the gasket in the centrifuge from being cut by the capillary tubes.

3. Insert the capillary tubes into the centrifuge with duplicate samples across from each other. Place the sealed

end toward the outside, making sure that the tubes are seated in the groves and firmly against the gasket.

4. If there is an internal cover, make sure it is screwed down. The tubes will break if the cover is not on correctly.

Centrifuge the specimens for five minutes at 11,000 to 12,000 rpms.

5. Open the centrifuge after it has come to a complete stop. Read the results immediately after the centrifuge

stops. If this is not possible, place the tubes upright until they are read. The red cells will slide if the tubes are left in

a horizontal position and the hematocrit will be falsely increased.

6. When reading hematocrits, make sure the clay red cell interface is aligned with the 0% line and the bottom of

the plasma meniscus is at the 100% line. The reading that corresponds to the top of the red cell column is the

hematocrit.

7. Duplicate readings should match within 1 L/L and must be within 2 L/L. Readings that match can be averaged

and reported in 0.5 L/L. Centrifuged hematocrits are always reported in whole numbers or halves.

Effect of Error on the

Hematocrit

Error Solution

Decreased Sample drawn above the IV line Redraw sample from another site

Decreased Red cell leakage Check sealant. Hard sealant causes

leaks.

Check gasket for cuts.

Do not fill and seal from the same end

of the capillary tube.

Decreased Short sample, red cells shrink Perform an automated hematocrit or

redraw the sample.

Decreased/Increased Mixing errors Mix sample well before each use.

Increased/Decreased Reading errors Check reading device by reading the

hematocrits on a card reader or with a

ruler.

Check employee competence.

Increased Time too short, timer not

working Inadequate rpms

Centrifuge longer, check timer.

Check centrifuge with tachometer.

Increased Sample allowed to stay in the

centrifuge after it has stopped

Remove samples immediately and

store them upright.

Increased Buffy coat included when

reading the red cell column

Carefully read the top of the red cell

column below the layer of white cells

and platelets.

Increased Abnormal red cell morphology

that results in increased trapped

plasma

Perform an automated hematocrit.

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Drawing of a centrifuged Hematocrit

Kathleen Kelly B.S., MT(ASCP)

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Hematocrit Tubes

Kathleen Kelly B.S., MT(ASCP)

Previous Page |

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Sealing a Hematocrit Tube

Kathleen Kelly B.S., MT(ASCP)

Filling and sealing with the clay perpendicular to the table keeps the clay from becoming contaminated with blood,

helps prevent leakage and is safer. Previous Page |

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Correctly Aligned Hematocrit Tube

Kathleen Kelly B.S., MT(ASCP)

The 100 % line must bisect the bottom of the meniscus and the zero line must align with the red cell/clay interface.

When both the 0% and 100% lines are positioned correctly, the hematocrit in per cent is the line that aligns with

the top of the red cells. The buffy coat, which is comprised of white cells and platelets, is not included. Previous

Page |

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a. Hematocrit centrifuge quality assurance

Kathleen Kelly B.S., MT(ASCP)

Quality assurance includes documented quality control and preventive maintenance plus the proficiency and

competency testing. Quality control Good quality control includes running duplicate samples on all patients and

performing at least two levels of commercial controls with known values. If clay is used to plug the tubes, it must

be soft, pliable, and inserted into the end that has not been coated with blood. Duplicate readings must be within

2% and are frequently within 1%.

Everyone performing hematocrits must demonstrate competency. An easy way to do this is to have everyone run

the same sample or a split sample and compare the results. This should include samples with normal and abnormal

hematocrits and be done on a quarterly basis. Quality assurance could also include documenting that multiple

people read the same spun hematocrits and obtained the same results. Proficiency testing samples are available

and may be combined with the proficiency testing done on a hematology multi parameter instrument.

Preventive maintenance

These are generic recommendations. Always check manufacturer's guidelines for specific instructions.

Daily:

Clean the inside of the centrifuge and the gasket.

Monthly:

Check the reading device. Misuse and zeroing of the reading devices can inject large errors. Always use a second,

simple reading device to check the fancier devices. Use a ruler or a flat plastic card. These cards are

available from laboratory vendors and are inexpensive.

Quarterly:

1. Check the brushes if they are present - this may need to be done more often.

2. Check the gasket for cuts and breaks. Cut gaskets allow tubes to leak and need to be replaced.

3. Check the timer with a stopwatch.

4. Check the speed of the centrifuge with a tachometer. These can easily be shared among multiple laboratories.

5. Perform a maximum cell pack to verify the time required for complete packing.

Centrifuge hematocrits (low, normal, high) for 2 minutes and then repeat the procedure adding 30 seconds each

time you centrifuge until the results are the same for two consecutive centrifugations. The required time is 30

seconds longer than the second time the hematocrit results match.

Example: The required time is 5 minutes.

Time 2 min. 2.5 min. 3 min. 3.5 min. 4 min. 4.5 min.

Result 26% 23% 22% 22%

Result 52% 49% 47% 46% 45% 45%

An easy way to check the maximum pack on a weekly basis is to read a sample and then recentrifuge it for one

minute. The results should be the same. If they are not, perform preventive maintenance on the centrifuge and

redo the maximum pack procedure.

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Micro-Hematocrit Capillary Tube reader

Kathleen Kelly B.S., MT(ASCP)

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3. Hemoglobins

Kathleen Kelly B.S., MT(ASCP)

Equipment that only performs hemoglobins is available. One of them is the HemoCue®. This small hand-held

equipment measures the hemoglobin in 10 microliters of blood. It measures azide methemoglobin by mixing

reagents and blood in a small disposable cuvet and measuring at two wavelengths. The higher wavelength

compensates for turbidity so lipemic samples are not a problem.

Stand-alone hemoglobinometers are also available. These are usually based on the same spectophotometric

principle as CBC analyzers and use cyanmethemoglobin as the end point.

With the inexpensive small multi parameter analyzers available, hemoglobin analyzers are no longer popular. The

use of the HemoCue® for back up and lipemic specimens is an exception.

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C. Red Cell and Platelet Indices

Kathleen Kelly B.S., MT(ASCP)

Red cell indices include the mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean

corpuscular hemoglobin content (MCHC) and red cell distribution width (RDW).

MCV is Mean Corpuscular Volume in femtoliters. On automated instruments, it is computed using the

measurements of each red cell. With manual methods, it is calculated using the hematocrit and the red cell count.

Hint: do not worry about the decimal points. This applies to the MCV, MCH and MCHC. Just divide the raw

numbers and place the decimal where it makes sense. It is a physiologic impossibility to have an MCV of 9.0 or 900.

It must be 90.

Manual

MCV =

spun hematocrit in L/L

------------------------------

red cells in millions/L -->

Automated Hematocrit = RBC x MCV

MCV correlates to the cell diameter on the peripheral smear. Macrocytes have a high MCV and

microcytes have a low MCV. It is possible to have abnormal sized cells and a normal MCV because the

MCV is an average. Agglutinated red cells also cause an increased MCV. These samples need to be

warmed in a 370 degrees C heating block and rerun warm.

MVC generic reference range: 82 - 99 femtoliters

MCH is Mean Corpuscular Hemoglobin weight in picograms. This is the average weight of the hemoglobin in

picograms in a red cell. It is a calculated value.

MCH = hemoglobin in gm/L

------------------------------

red cell count in millions/L

MCH generic reference range: 27 - 32 picograms

MCHC is Mean Corpuscular Hemoglobin Content. This indicates the average weight of hemoglobin as compared to

the cell size. It is traditionally a calculated value. How ever some instruments may measure the density of the cells

as they are counted and use this value to compare to the calculated value. Bayer calls this measured value a CHCM

(Cellular Hemoglobin Concentration Mean) on the Bayer/Technicon hematology instruments.

MCHC

=

Hemoglobin in g/mL

------------------------------

Red cell count in millions/L

or

MCHC

=

MCH in picograms

-------------------------------

MCV in femtoliters

MCHC correlates with the degree of hemoglobinization of the red cells on the peripheral smear.

It is reported in gm/dL, picograms/100 femtoliter or in %. A decreased MCHC corresponds to cells with increased

zones of central pallor on a Wright's stained peripheral smear. These cells are called hypochromic red cells.

An increased MCHC is rarely a true value. MCHCs above the reference range are suspect. A large number of

spherocytes, the most common physiologic reason, is not common. A more likely reason is an error in the

hemoglobin or the hematocrit. Solutions include: checking the smear for spherocytes, retesting the sample,

performing a spun microhematocrit, performing an alternate hemoglobin method, and checking the quality control

and other patient results to identify shifts or trends in the hemoglobin or hematocrit determinations.

MCHC generic reference range: 32-36 g/dL or pg/fL

RDW is the Red cell Distribution Width. This value indicates the degree of red cell size variation or how much

difference exists between the largest and smallest red cells. This value is derived from the MCV histogram. An

increased RDW corresponds with an increase in anisocytosis on the peripheral smear. The RDW is

only available if it is included in the instrument menu. Although different manufacturers use slightly different

methods of obtaining data the RDW is generally thought of as the coefficient of variation of red cell volume

distribution.

RDW = standard deviation x100

----------------------

mean MCV

RDW generic reference range: 9.0 - 14.5

The RDW, coupled with the MCV, gives more relevant information than an individual index. The following is an

attempt to clarify the relationship of the MCV and RDW.

1. Red cells that are all microcytic or macrocytic will have a RDW in the reference range and a decreased or an

increased MCV.

2. Red cells that vary in size and have an average size within the reference range will have an increased RDW

and a normal MCV.

3. Red cells that vary in size and have an average size below or above the reference range will have an

abnormal MCV and RDW.

There are varying opinions on the clinical value of red cell indices. They are used to morphologically classify

anemias and to select additional tests to determine the cause of an anemia. Indices also change in response to

treatment of some anemias.

Platelet Indices: These include the platelet distribution width and average platelet volume. They have limited use,

but do correlate to flags on the platelet histogram that indicate giant platelet or platelet clumps.

Anisocytosis - variance in red cell size as determined by viewing the diameter of the cells on a stained peripheral

blood smear.

Anemia - A reduction in the oxygen carrying capacity of the blood. Almost always manifested by a decreased

hemoglobin and frequently accompanied by a decreased hematocrit and red cell count.

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Macrocytic Red Cells

Kathleen Kelly B.S., MT(ASCP)

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Microcytic Red Cells

Kathleen Kelly B.S., MT(ASCP)

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Macrocytic and Microcytic Red Cells

Kathleen Kelly B.S., MT(ASCP)

These red cells vary in size and have an average cell size that falls with in the reference range. Previous Page |

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Hypochromic Red Cells

Kathleen Kelly B.S., MT(ASCP)

Hypochromic red cells have an increased zone of pallor on Wright's stain. These cells contain decreased

hemoglobin for their size and have a decreased MCHC. Previous Page |

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Anisocytosis: Variation in Red Cell Size

Kathleen Kelly B.S., MT(ASCP)

These cells exhibit a large variation in size. The RDW (Red Cell Distribution width) is a numerical indication of this

morphologic abnormality. Previous Page |

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