Beth Israel Deaconess Medical Center Laboratory Manual

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The physicians, nurses, and other professionals who take care of neonates frequently ask about strategies on how to use as little blood as possible for their patients in whom common sample volumes may represent a significant proportion of their blood volume.  In this section we list the tests most frequently ordered on neonates, specimen requirements, and minimum volumes.

One strategy that can be used to great advantage is to order more tests at one time on one sample rather than ordering one or two tests on many samples.  Because of the way automation works, it is much more efficient to do as many tests as possible at one time.

Thus, for example, if you need a gentamicin level at a specific time, it is wise to order the electrolytes, CK, and magnesium at the same time.  Ordered this way, you will only need a single microtainer tube.  If, however, you order the gentamicin on one sample and then the other tests later (or even if you try to “add on” the other tests to the gentamicin sample), it will take twice as much blood.

Similarly, for coagulation studies (PT, PTT, fibrinogen), one must always FILL the blue top “pedi” tube, so that the proper amount of anticoagulant is present (i.e., underfilling will give erroneous results).  Thus, it makes sense to order all the necessary tests at once rather than ordering each test at separate times (which would require a “full” tube each time).  The patient’s hematocrit is also an important aspect of proper specimen collection for blue top tubes.  If the patient’s Hct is > 55%, the amount of anticoagulant in the tube must be adjusted for accurate coagulation determinations.  Call the Clinical Laboratory (ask for a hematology supervisor) prior to specimen collection.

Some other notes about volumes are important.  As noted above, one must always FILL blue top  tubes appropriately (a “pedi” blue top holds 2.7 mL, minimum acceptable is 2.0 mL).  For purple top “microtainer” tubes, one must use a minimum of 0.5 mL (500 μl); filled they hold 0.75 mL (750 μl). 
A plasma separator tube is recommended for routine Chemistries.  Most routine Chemistry tests can be performed on  plasma as well as serum.  A well mixed plasma separator microtainer will eliminate the clotty sample issue that we see with serum separator microtainers..  Eliminating the clotty samples will reduce TAT as well as reduce the number of QNS samples due to clotty serum.  For plasma separator and serum separator “microtainer” tubes, the patient’s hematocrit is also important:  these tubes are centrifuged to separate the red blood cells from the plasma/serum, which is what we analyze.    In the table below, we have assumed a Hct of 50%; if it’s much higher, you’ll need to draw proportionately more blood.  A filled plasma separator or serum separator microtainer (to top line) holds 0.7 mL (700 μ) of blood, which would yield (at Hct=50%), roughly 0.35 mL (350 μl) of plasma/serum.  A plasma separator or serum separator tube filed to the second line from the top holds 0.4ml (400 μl) blood.  As you fill the microtainer, tap gently on a flat surface to displace air bubbles in the tube to achieve the appropriate fill.

If you use the absolute minimums listed here (especially for chemistry tests), there may not be sufficient residual volumes to do confirmations, dilutions, etc.

For the availability of tests and turnaround times, please consult the individual sections of this lab manual.  This section is devoted exclusively to special volume requirements.  As always, if you have questions about tests (availability, turnaround time, specimen requirements) that are not addressed here, ask to speak to a supervisor in the laboratory.

Neonatal Specimen Requirements for Commonly Ordered Tests

TEST	CONTAINER	MINIMUM VOLUME	COMMENTS
Blood Bank
Type and Direct Antiglobulin Test (DAT, Coombs)	purple "pedi" tube	1 ml
Type and Crossmatch	purple "pedi" tube	1 ml	Unexpected serological problems will require additional sample.
Chemistry
ABGs alone	1 ml low heparin syringe	0.100 ml	Fill to 0.1 line.
ABGs in combination with electrolytes, ionized Ca, or Hct	1 ml low heparin syringe	0.200 ml	Fill to 0.2 line.
Automated Chemistry Tests (Gluc, BUN, Creat, Lytes, LFT’s, Ca, Mg, Trig, Alb, Phos)	Plasma separator or serum separator microtainer	0.4 ml (single test) 0.7 ml (any combination)	Fill to 2nd line from the top. Fill to top line.
Bilirubin, Total & Direct	Plasma separator or serum separator microtainer	0.4 ml	Fill to 2nd line from the top.
Glucose	Plasma separator or serum separator microtainer	0.4 ml	Fill to 2nd line from the top.
Gentamicin or Vancomycin	Plasma separator or serum separator microtainer	0.4 ml	Fill to 2nd line from the top. If ordered with any automated chemistries, send a full tube.
Theophylline or Phenobarbital	Plasma separator or serum separator microtainer	0.4 ml	Fill to 2nd line from the top. If ordered with any automated chemistries, send a full tube.
Thyroid Function Tests (TSH, T4, T3, T3 uptake)	Plasma separator or serum separator microtainer	1.4 ml	Two full tubes required for all 4 tests. One tube required for TSH only. For each additional test, add 1/2 tube.
Ammonia	Plasma separator  microtainer	0.4 ml	Fill to 400 line.
Cortisol	Plasma separator or serum separator microtainer	0.4 ml	Fill to 2nd line from the top.
FSH	Plasma separator or serum separator microtainer	0.4 ml	Fill to 2nd line from the top.
LH	Plasma separator or serum separator microtainer	0.4 ml	Fill to 2nd line from the top.
Prolactin	Plasma separator or serum separator microtainer	0.4 ml	Fill to 2nd line from the top.
Testosterone	Plasma separator or serum separator microtainer	0.4 ml	Fill to 2nd line from the top.
Toxicology Screen (urine)	urine container	5 ml urine	1 ml sufficient for in-house screen only. Additional sample needed if confirmation required.
Urine Electrolytes	urine container	0.5 ml urine
CSF Total protein, glucose	sterile tube #2 or #3 from CSF collection kit	0.2 ml
Cytogenetics
Chromosome Analysis	green top "pedi" tube	1.5 ml blood

Neonatal Specimen Requirements for Commonly Ordered Tests (continued)

Hematology
CBC (with or without differential)	purple top microtainer	0.5 ml	Fill to 500 line. CBC includes platelet count. These specimen requirements pertain to any part of the CBC. (e.g. even if only Hct or Plt Ct ordered).
G6PD	purple top microtainer	0.75 ml	Fill to 750 line. If Reticulocyte >5%, 1 "pedi" purple top (2.7ml) required.
Reticulocyte	purple top microtainer	0.5 ml	Fill to 500 line. If ordered in addition to CBC, 0.75ml required (fill to 750 line).
PT, PTT,and/or Fibrinogen	blue top "pedi" tube	2 ml	1 Full Tube (2.7ml draw preferred). If any of these tests are ordered individually, same amounts required. Any other coagulation tests added to one of these requests (e.g. D-Dimer, Heparin) requires an additional tube.
FDP	special FDP tube	2ml (do not overfill)	Call lab for tube if not available.
CSF cell count and diff	sterile tube #4 and #1 from CSF collection kit	0.2 ml	If 4 tubes are not collected, send last collected tube for cell count/diff.
Microbiology
Blood Culture (aerobic)	Peds Plus blood culture bottle	0.5 ml	1-3 ml blood preferred for aerobic.  5 ml maximum.
Blood Culture (anaerobic)	Lytic/10 anaerobic blood culture bottles	3 ml	Anaerobic blood culture by request only.
Urine Culture	sterile container	0.1 ml	1 ml preferred
CSF Culture	sterile tube #2 or #3 from CSF collection kit	0.1 ml	1 ml preferred
CMV (urine)	sterile container	1.0 ml	Must be sent on ice.  1.5 ml preferred.
Herpes Simplex Virus (CSF) for PCR	sterile tube #2 or #3 from CSF collection kit	0.5 ml	PCR more sensitive than culture.
Viral Culture (CSF)	sterile tube #2 or #3 from CSF collection kit	0.6 ml	Transport on ice in viral transport medium (M4-RT) and deliver immediately to the Microbiology Lab.  1 ml preferred.
VDRL (CSF)	sterile tube #2 or #3 from CSF collection kit	0.5 ml
Miscellaneous
1,25 dihydroxy Vitamin D	red top pedi tube	1.1 ml	3 mL
11-Deoxycortisol	serum separator	0.2 ml	Fill to top line
17-OH Pregnenelone	serum separator	0.4 ml	Fill 2 tubes
17-OH Progesterone	serum separator	0.1 ml	Fill to second line from top
21-Hydroxylase deficiency	EDTA pedi tube	5 ml	Fill 2 tubes
25-OH Vitamin D	serum separator	0.4 ml	Fill 2 tubes
7-Dehydrocholesterol	purple top microtainer	0.1 ml	Kennedy Krieger Form
8(9)-Cholestenol	EDTA pedi tube	3 ml	Kennedy Krieger Form
ACTH	EDTA pedi tube	1 ml	Send immediately to the lab
Acylcarnitine Profile	Green top pedi tube	1 ml	Send immediately to the lab
Acylglycines	Urine	3 ml
ADH	EDTA pedi tube	3 ml	Tube must be prechilled
Aldosterone	serum separator	0.2 ml	Fill to top line
Amino Acid	CSF	0.5 ml
Amino Acid (plasma)	green top "pedi" tube	1 ml	Must be sent to lab immediately for special processing.
Amino Acid (urine)	urine container	2.2 ml
Androstenedione	serum separator	0.5 ml	Fill 2 tubes
Anti-TPO	serum separator	0.2 ml	Fill to top line
Beta-hydroxybutyrate	serum separator	0.5 ml	Fill 2 tubes
Calcitonin	serum separator	0.3 ml	Fill to top line
Cholestanol	EDTA pedi tube	3 ml	Kennedy Krieger Form
C-peptide	serum separator	0.4 ml	Fill 2 tubes
DHEA	serum separator	0.3 ml	Fill to top line
Estrogen	serum separator	0.5 ml	Fill 2 tubes
Free Fatty Acids	serum separator	0.2 ml	Fill to top line; send immediately to lab
Glucagon	red top pedi tube	1.1 ml	3 ml
Growth Hormone	serum separator microtainer	0.7 ml	Fill to top line.
IGF-1     (Somatomedin C)	serum separator	0.4 ml	Fill 2 tubes
Insulin	serum separator	0.4 ml	Fill 2 tubes
Iodine	urine	2 ml
Lactate/Pyruvate	gray top "pedi" tube	3.0 ml	Tube must be pre-chilled.
Organic Acid (plasma)	green top "pedi" tube	1 ml
Organic Acid (urine)	urine container	5 ml
Orotic Acid	urine	10 ml
Pipecolic Acid	EDTA pedi tube	3 ml	Kennedy Krieger Form
Pipecolic Acid	urine	10 ml	Kennedy Krieger Form
Plasma Very Long Chain Fatty Acids	EDTA pedi tube	3 ml	Kennedy Krieger Form
Prader Willi Syndrome	EDTA pedi tube	3 ml	*BU Center of Human Genetics Form
Proinsulin	serum separator	0.5 ml	Fill 2 tubes
PTHRP	Call lab	6 ml	Special tube - stored frozen in lab
Pyruvate	CSF	0.5 ml
Red Blood Cell Plasmalogen	EDTA pedi tube	3 ml	Kennedy Krieger Form
Reducing Substances	urine	1 ml
Reverse T3	serum separator	0.4 ml	Fill 2 tubes
Subtelomeric FISH	Green top pedi tube	3 ml
TSH binding inhibitory antibody	serum separator	0.2 ml	Fill to top line
TSI	serum separator	0.4 ml	Fill 2 tubes